本發(fā)明涉及一種具有多個(gè)SV40核定位序列的SpyCas9蛋白及其應(yīng)用。

背景技術(shù)：

CRISPR/Cas9技術(shù)是近兩年出現(xiàn)的基因編輯技術(shù)，因其使用方便的特點(diǎn)在世界范圍內(nèi)得到廣泛應(yīng)用。其發(fā)明專利所屬目前還有爭(zhēng)議，Broad研究所(Broad Institute of MIT and Harvard)和加州大學(xué)伯克利分校在此專利上還在進(jìn)行訴訟。加州大學(xué)伯克利分校的Jennifer Doudna課題組第一次報(bào)道CRISPR/Cas9可用于特異性的DNA切割(文獻(xiàn)1)。隨后美國(guó)Broad研究所的Feng Zhang實(shí)驗(yàn)室(文獻(xiàn)2)以及George Church實(shí)驗(yàn)室(文獻(xiàn)3)以及韓國(guó)首爾大學(xué)的Jin-Soo Kim課題組(文獻(xiàn)4)報(bào)道了此技術(shù)應(yīng)用于人體細(xì)胞基因組的改造。

在CRISPR/Cas9系統(tǒng)發(fā)揮作用時(shí)，需要將編碼Cas9核酸酶以及單鏈導(dǎo)引RNA(sgRNA)的DNA或RNA序列通過(guò)脂質(zhì)體轉(zhuǎn)染、電擊、顯微注射等方式運(yùn)輸?shù)郊?xì)胞中。韓國(guó)首爾大學(xué)的Jin-Soo Kim課題組(文獻(xiàn)5)以及韓國(guó)漢陽(yáng)大學(xué)的Hyongbum Kim課題組(文獻(xiàn)6)證明了可以將Cas9核酸酶在體外(如大腸桿菌中)進(jìn)行表達(dá)純化，與體外轉(zhuǎn)錄的sgRNA的RNA進(jìn)行混合，形成核酸蛋白復(fù)合物(ribonucleoprotein，RNP)，將RNP通過(guò)核轉(zhuǎn)染(電擊)等方式運(yùn)輸?shù)郊?xì)胞中，從而對(duì)細(xì)胞基因組進(jìn)行改造。

參考文獻(xiàn)

1.Jinek，M.，Chylinski，K.，F(xiàn)onfara，I.，Hauer，M.，Doudna，J.A.and Charpentier，E.(2012)A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science，337，816-821.

2.Cong，L.，Ran，F(xiàn).A.，Cox，D.，Lin，S.，Barretto，R.，Habib，N.，Hsu，P.D.，Wu，X.，Jiang，W.，Marraffini，L.A.et al.(2013)Multiplex genome engineering using CRISPR/Cas systems.Science，339，819-823.

3.Mali，P.，Yang，L.，Esvelt，K.M.，Aach，J.，Guell，M.，DiCarlo，J.E.，Norville，J.E.and Church，G.M.(2013)RNA-guided human genome engineering via Cas9.Science，339，823-826.

4.Cho，S.W.，Kim，S.，Kim，J.M.and Kim，J.S.(2013)Targeted genome engineering in human cells with the Cas9RNA-guided endonuclease.Nat.Biotechnol.，31,230-232.

5.Kim，S.，Kim，D.，Cho，S.W.，Kim，J.and Kim，J.S.(2014)Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9ribonucleoproteins.Genome Res.，24，1012-1019.

6.Ramakrishna，S.，Kwaku Dad，A.B.，Beloor，J.，Gopalappa，R.，Lee，S.K.and Kim，H.(2014)Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA.Genome Res.，24，1020-1027.

7.Liu，J.，Gaj，T.，Yang，Y.，Wang，N.，Shui，S.，Kim，S.，Kanchiswamy，C.N.，Kim，J.S.and Barbas，C.F.，3rd.(2015)Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells.Nat.Protoc.，10，1842-1859.

技術(shù)實(shí)現(xiàn)要素：

本發(fā)明的目的是對(duì)SpyCas9核酸酶蛋白質(zhì)進(jìn)行改進(jìn)，提高其基因編輯效率。

為了達(dá)到上述目的，本發(fā)明提供了一種具有多個(gè)核定位序列的SpyCas9蛋白，其特征在于，包括SpyCas9蛋白，其中，所述的SpyCas9蛋白的氨基端和/或羧基端具有至少2個(gè)SV40核定位序列(Nuclear localization signal，簡(jiǎn)稱NLS)。

優(yōu)選地，所述的SV40核定位序列為PKKKRKV。

優(yōu)選地，所述的SpyCas9蛋白的氨基端和羧基端兩端含有共計(jì)3個(gè)及以上的SV40核定位序列。

優(yōu)選地，所述的具有多個(gè)核定位序列的SpyCas9蛋白的序列為SEQ ID NO：14～SEQ ID NO：24中的一種。

本發(fā)明還提供了上述的具有多個(gè)核定位序列的SpyCas9蛋白在基因編輯中的應(yīng)用。

優(yōu)選地，所述的基因編輯的運(yùn)輸方式為核轉(zhuǎn)染、脂質(zhì)體轉(zhuǎn)染或顯微注射。

優(yōu)選地，所述的基因編輯為對(duì)于細(xì)胞系、原代細(xì)胞、前體細(xì)胞或干細(xì)胞的基因編輯。

一種基因編輯試劑盒，其特征在于，含有上述的具有多個(gè)核定位序列的SpyCas9蛋白。

與現(xiàn)有技術(shù)相比，本發(fā)明的有益效果：

1、如圖1所示，本發(fā)明通過(guò)對(duì)SpyCas9核酸酶蛋白質(zhì)的氨基端及羧基端增加SV40核定位序列來(lái)增加其基因編輯效率。

2、采用核轉(zhuǎn)染、脂質(zhì)體轉(zhuǎn)染和顯微注射等運(yùn)輸方式，本發(fā)明的具有多個(gè)核定位序列的SpyCas9蛋白，尤其是所述的SpyCas9蛋白的氨基端或羧基端一端含有至少2個(gè)SV40核定位序列以及所述的SpyCas9蛋白的氨基端和羧基端兩端含有共計(jì)3個(gè)及以上的SV40核定位序列，可以比傳統(tǒng)的SpyCas9(氨基或羧基端含有1個(gè)NLS序列)具有更高的基因編輯效率；

3、本發(fā)明的具有多個(gè)核定位序列的SpyCas9蛋白對(duì)細(xì)胞系，也適用于原代細(xì)胞、前體細(xì)胞以及干細(xì)胞均具有更高的基因編輯效率。

附圖說(shuō)明

圖1為具有多個(gè)核定位序列的SpyCas9蛋白設(shè)計(jì)圖；在SpyCas9核酸酶蛋白質(zhì)的羧基端或氨基端引入多個(gè)SV40NLS(氨基酸序列為PKKKRKV)。

圖2為核轉(zhuǎn)染結(jié)果圖；多個(gè)NLS改造可以提高以核轉(zhuǎn)染方式運(yùn)輸?shù)腟pyCas9蛋白質(zhì)在人K562細(xì)胞系、人間充質(zhì)干細(xì)胞(mesenchymal stem cells)、人原代成纖維細(xì)胞(human dermal fibroblast)以及人神經(jīng)前體細(xì)胞(Neural progenitor cells)上的基因編輯效率。通過(guò)對(duì)基因編輯的細(xì)胞進(jìn)行T7E1酶切處理(文獻(xiàn)7)可以檢測(cè)基因編輯效率，箭頭所指為T7E1酶切割產(chǎn)物。負(fù)對(duì)照為用不含有SpyCas9蛋白質(zhì)的緩沖液進(jìn)行處理。命名方式為：N代表氨基端，C代表羧基端，數(shù)字代表NLS個(gè)數(shù)(下同)。具體命名與序列ID對(duì)應(yīng)關(guān)系為：N0/C0(SEQ ID：10)，N1/C0(SEQ ID：11)，N0/C1(SEQ ID：12)，N1/C1(SEQ ID：13)，N0/C2(SEQ ID：14)，N0/C3(SEQ ID：15)，N0/C4(SEQ ID：16)，N1/C2(SEQ ID：17)，N1/C3(SEQ ID：18)，N2/C0(SEQ ID：19)，N2/C1(SEQ ID：20)，N2/C2(SEQ ID：21)，N3/C0(SEQ ID：22)，N3/C1(SEQ ID：23)，N4/C0(SEQ ID：24)。

圖3為脂質(zhì)體轉(zhuǎn)染結(jié)果圖；多個(gè)NLS改造可以提高SpyCas9蛋白質(zhì)以脂質(zhì)體細(xì)胞運(yùn)輸方式進(jìn)行的基因編輯效率。通過(guò)實(shí)驗(yàn)室自主構(gòu)建的EGFP報(bào)告細(xì)胞系(基于HEK293T)，對(duì)多NLS的SpyCas9蛋白質(zhì)切割DNA、激活EGFP的效率進(jìn)行衡量。樣品從左到右分別為：負(fù)對(duì)照、N0/C0(SEQ ID：10)、N0/C1(SEQ ID：12)、N0/C2(SEQ ID：14)、N0/C3(SEQ ID：15)、N0/C4(SEQ ID：16)、N1/C0(SEQ ID：11)、N1/C1(SEQ ID：13)、N1/C2(SEQ ID：17)、N1/C3(SEQ ID：18)、N2/C0(SEQ ID：19)、N2/C1(SEQ ID：20)、N2/C2(SEQ ID：21)、N3/C0(SEQ ID：22)、N3/C1(SEQ ID：23)以及N4/C0(SEQ ID：24)。

圖4為顯微注射結(jié)果圖。多個(gè)NLS改造可以提高SpyCas9蛋白質(zhì)以顯微注射方式進(jìn)行的基因編輯效率。將傳統(tǒng)和NLS改造的SpyCas9蛋白質(zhì)分別與sgRNA混合，注射進(jìn)入小鼠胚胎，然后對(duì)第三天8細(xì)胞期的胚胎進(jìn)行PCR擴(kuò)增，測(cè)序鑒定突變，發(fā)現(xiàn)多個(gè)NLS顯著提高對(duì)PTEN的基因編輯效率。

具體實(shí)施方式

下面結(jié)合具體實(shí)施例，進(jìn)一步闡述本發(fā)明。應(yīng)理解，這些實(shí)施例僅用于說(shuō)明本發(fā)明而不用于限制本發(fā)明的范圍。此外應(yīng)理解，在閱讀了本發(fā)明講授的內(nèi)容之后，本領(lǐng)域技術(shù)人員可以對(duì)本發(fā)明作各種改動(dòng)或修改，這些等價(jià)形式同樣落于本申請(qǐng)所附權(quán)利要求書所限定的范圍。

實(shí)施例1核轉(zhuǎn)染方式的細(xì)胞運(yùn)輸

本實(shí)施例將純化的含有多個(gè)SV40NLS序列的SpyCas9蛋白質(zhì)，與體外轉(zhuǎn)錄的sgRNA共同孵育，形成核酸蛋白復(fù)合物(RNP)，與細(xì)胞懸浮液混合后，通過(guò)核轉(zhuǎn)染儀電擊，將RNP運(yùn)輸?shù)郊?xì)胞中。實(shí)驗(yàn)證明，多個(gè)NLS的SpyCas9比傳統(tǒng)SpyCas9具有更高的基因編輯效率。

1實(shí)驗(yàn)方法：

1)K562細(xì)胞的準(zhǔn)備。將K562細(xì)胞(ATCC，CCL-243)培養(yǎng)在RPMI1640培養(yǎng)基(賽默飛公司，貨號(hào)61870150)中，添加10％的小牛血清(賽默飛，10099141)以及100單位/毫升的penicillin和100單位/毫升的streptomycin(賽默飛，15140122)，在37℃、5％二氧化碳、飽和濕度的恒溫培養(yǎng)箱中培養(yǎng)24小時(shí)。將2×10⁵個(gè)細(xì)胞離心去上清，重懸于20微升P3核轉(zhuǎn)染液(Lonza，V4XP-3012)中。

2)人間充質(zhì)干細(xì)胞的準(zhǔn)備。將人間充質(zhì)干細(xì)胞(ATCC，PCS-500-012)培養(yǎng)在鋪有Matrigel(BD Bioscience，354234)的MesenPRO RS培養(yǎng)基(賽默飛，12746012)中，在37℃、5％二氧化碳、飽和濕度的恒溫培養(yǎng)箱中培養(yǎng)24小時(shí)。將細(xì)胞用Accutase酶(StemCell Technologies，07920)在37℃處理1分鐘，用MesenPRO RS完全培養(yǎng)基中和后離心去上清，取出2×10⁵個(gè)細(xì)胞重懸于20微升P3核轉(zhuǎn)染液(Lonza，V4XP-3012)。

3)人原代成纖維細(xì)胞的準(zhǔn)備。將人原代成纖維細(xì)胞(賽默飛，C0135C)培養(yǎng)在DMEM培養(yǎng)基(賽默飛公司，貨號(hào)10566-016)中，添加10％的小牛血清(賽默飛，10099141)以及100單位/毫升的penicillin和100單位/毫升的streptomycin(賽默飛，15140122)，在37℃、5％二氧化碳、飽和濕度的恒溫培養(yǎng)箱中培養(yǎng)24小時(shí)。將細(xì)胞用胰酶(賽默飛，25300054)在37℃處理1分鐘，用DMEM+10％小牛血清完全培養(yǎng)基中和后離心去上清，取出2×10⁵個(gè)細(xì)胞重懸于20微升P3核轉(zhuǎn)染液(Lonza，V4XP-3012)。

4)人神經(jīng)前體細(xì)胞的準(zhǔn)備。將人神經(jīng)前體細(xì)胞(ATCC，ACS-5003)培養(yǎng)在鋪有Matrigel(BD Bioscience，354234)的擴(kuò)增培養(yǎng)基(Lonza，CC-4241)中，添加了1×培養(yǎng)基添加物(Lonza，CC-4242)，在37℃、5％二氧化碳、飽和濕度的恒溫培養(yǎng)箱中培養(yǎng)24小時(shí)。將2×10⁵個(gè)細(xì)胞用Accutase酶(StemCell Technologies，07920)在37℃處理1分鐘，用完全培養(yǎng)基中和后離心去上清，取出2×10⁵個(gè)細(xì)胞重懸于20微升P3核轉(zhuǎn)染液(Lonza，V4XP-3012)。

5)SpyCas9蛋白質(zhì)的表達(dá)與純化。從金唯智(南京)采用常規(guī)的基因合成方法合成含有SpyCas9基因的PCR DNA(金唯智提供；SEQ ID：10～SEQ ID：24)，克隆進(jìn)入pET28b載體(Novogene，69865-3)。具體步驟如下：1.酶切：在50微升反應(yīng)體系為：10微克含有SpyCas9基因的PCR DNA(金唯智提供；SEQ ID：10～SEQ ID：24)、1微升XbaI限制性內(nèi)切酶(NEB，R0145S)、1微升Xho I限制性內(nèi)切酶(NEB，R0146S)、1×NEB buffer 4(NEB，B7004S)，加水補(bǔ)足50微升。在37℃處理4小時(shí)。對(duì)pET28b載體同樣處理。用膠回收試劑盒(Qiagen，28704)分別對(duì)SpyCas9基因和pET28b目的片段進(jìn)行回收。2.連接。10微升反應(yīng)體系為：含有50微克純化的pET28b酶切載體、25微克純化的SpyCas9基因(SEQ ID：14～SEQ ID：24)、1微升T4DNA連接酶(NEB，M0202S)，1×T4 DNA連接酶緩沖液(NEB，B0202S)，加水補(bǔ)足10微升。在16℃反應(yīng)過(guò)夜，得到連接產(chǎn)物。3.轉(zhuǎn)化。將5微升連接產(chǎn)物按照說(shuō)明轉(zhuǎn)化進(jìn)入Trans10化學(xué)轉(zhuǎn)化感受態(tài)細(xì)胞(全式金(CD101-01))。將轉(zhuǎn)化獲得克隆送往金唯智采用常規(guī)方法提取質(zhì)粒并進(jìn)行測(cè)序驗(yàn)證序列(金唯智)。將pET28b-SpyCas9質(zhì)粒按照試劑盒說(shuō)明轉(zhuǎn)化進(jìn)入BL21(DE3)細(xì)胞(賽默飛，C600003)，采用1升含有50微克/毫升卡那霉素(Sigma-Aldrich，K0100000)抗性的LB培養(yǎng)基(賽默飛，10855021)搖床上37℃進(jìn)行培養(yǎng)至OD₆₀₀達(dá)到0.8，用0.1mM的IPTG(Sigma-Aldrich，I6758-1G)誘導(dǎo)表達(dá)，16℃培養(yǎng)過(guò)夜。第二天，收集菌體，重懸于30毫升裂解緩沖液(20mM TrisHCl(Sigma-Aldrich，000000010708976001)，pH 8.0，0.5M NaCl(Sigma-Aldrich，S3014-500G))，再添加1mM TCEP(Sigma-Aldrich，000000000000024153)和1×蛋白酶抑制劑(羅氏，SRE0055)。通過(guò)超聲波對(duì)細(xì)胞進(jìn)行破碎，離心取上清，加入Ni-NTA瓊脂糖珠子(Qiagen，30210)結(jié)合含有His標(biāo)簽的SpyCas9蛋白質(zhì)。用裂解緩沖液沖洗結(jié)合有SpyCas9蛋白質(zhì)的Ni-NTA珠子，用含有300mM咪唑(Sigma-Aldrich，792527-100G)的裂解緩沖液洗脫結(jié)合的SpyCas9蛋白質(zhì)。

6)體外轉(zhuǎn)錄sgRNA。以pU6-sgRNA質(zhì)粒(Addgene，64245)為模板，用含有T7啟動(dòng)子及目的位點(diǎn)序列的引物(金唯智，南京)進(jìn)行PCR，擴(kuò)增針對(duì)CCR5、EGFP以PTEN的sgRNA序列(50μL的PCR溶液：1μL質(zhì)粒模板、1×Taq Master Mix(NEB，M0270L)，0.2μM的引物組合sgRNACCR5-F(SEQ ID：2)/sgRNA-R(SEQ ID：5)、sgRNAEGFP-F(SEQ ID：3)/sgRNA-R(SEQ ID：5)或是sgRNAPTEN-F(SEQ ID：4)/sgRNA-R(SEQ ID：5)，加水補(bǔ)足50μL，分別用來(lái)擴(kuò)增對(duì)CCR5、EGFP及PTEN的sgRNA；PCR條件為：95℃10分鐘，40個(gè)循環(huán)的95℃30秒、55℃30秒以及72℃1分鐘，最后加上72℃10分鐘))，并用瓊脂糖凝膠電泳純化PCR產(chǎn)物。采用HiScibe T7 Quick High yield RNA Synthesis kit試劑盒(NEB，E2050)以PCR產(chǎn)物為模板，按照試劑盒操作說(shuō)明合成獲得sgRNA。

7)SpyCas9-sgRNA核酸蛋白質(zhì)復(fù)合物(RNP)的準(zhǔn)備。將30微克純化的SpyCas9蛋白質(zhì)與30微克純化的sgRNA混合，室溫孵育10分鐘。

8)核轉(zhuǎn)染儀點(diǎn)擊實(shí)驗(yàn)。將1微升SpyCas9RNP(30微克SpyCas9蛋白與30微克sgRNA)加入20微升如上幾種細(xì)胞重懸液，充分混合后加入8聯(lián)排點(diǎn)擊管，在4D Nucleofector核轉(zhuǎn)染儀器(Lonza，AAF-1002B和AAF-1002F)上選取每個(gè)細(xì)胞類型預(yù)設(shè)定的程序進(jìn)行電擊，然后將細(xì)胞分別按照如上培養(yǎng)條件接種培養(yǎng)皿中進(jìn)行培養(yǎng)。48小時(shí)后，收取細(xì)胞樣品，對(duì)基因編輯效率進(jìn)行分析(如下)。

9)通過(guò)T7E1檢測(cè)基因編輯效率(文獻(xiàn)7)。收集如上所述處理過(guò)的細(xì)胞，用50微升的QuickExtract(Epicentre，QEB0905T)緩沖液裂解，用引物CCR5-F(SEQ ID：6)和CCR5-R(SEQ ID：7)對(duì)細(xì)胞人CCR5基因進(jìn)行PCR擴(kuò)增(50μL的PCR溶液：1微升QuickExtract基因組裂解液、1×Taq Master Mix(NEB，M0270L)，0.2μM的引物CCR5-F和CCR5-R，加水補(bǔ)足50μL；PCR條件為：95℃10分鐘，40個(gè)循環(huán)的95℃30秒、55℃30秒以及72℃1分鐘，最后加上72℃10分鐘)。將PCR產(chǎn)物加熱至95℃，隨后緩慢降溫直至室溫。取出400ng PCR產(chǎn)物DNA，加入1微升T7E1核酸內(nèi)切酶(NEB，M0302L)，37℃孵育1小時(shí)。將酶切產(chǎn)物進(jìn)行瓊脂糖凝膠電泳，獲得附圖2結(jié)果。

2結(jié)果分析：

下面比較了本發(fā)明的具有多個(gè)核定位序列的SpyCas9和傳統(tǒng)SpyCas9的差別。為命名方便，氨基端(N端)和羧基端(C端)的NLS分別用數(shù)字表示，如N0/C2表示氨基端含有0個(gè)NLS、羧基端含有2個(gè)NLS的SpyCas9。

1)人K562細(xì)胞T7E1檢測(cè)結(jié)果。通過(guò)比較發(fā)現(xiàn)，具有多個(gè)核定位序列的SpyCas9的基因編輯效率顯著提升，如N0/C2、N0/C3、N0/C4相比N0/C1(傳統(tǒng))的SpyCas9效率均有提升。

2)人間充質(zhì)干細(xì)胞T7E1檢測(cè)結(jié)果。通過(guò)比較發(fā)現(xiàn)，在N0/C1(傳統(tǒng))SpyCas9無(wú)法實(shí)現(xiàn)基因編輯的情況下，具有多個(gè)核定位序列的SpyCas9(如N0/C2及N0/C4)能成功實(shí)現(xiàn)基因編輯。

3)人原代成纖維細(xì)胞。通過(guò)比較發(fā)現(xiàn)，N0/C2、N0/C3、N0/C4相比N0/C1(傳統(tǒng))的SpyCas9效率均有提升。

4)人神經(jīng)前體細(xì)胞。通過(guò)比較發(fā)現(xiàn)，N0/C2、N0/C3、N0/C4相比N0/C1(傳統(tǒng))的SpyCas9效率均有提升。

實(shí)施例2脂質(zhì)體方式的細(xì)胞運(yùn)輸

本實(shí)施例將純化的具有多個(gè)核定位序列的SpyCas9蛋白，與體外轉(zhuǎn)錄的sgRNA共同孵育，形成核酸蛋白復(fù)合物(RNP)，再與脂質(zhì)體(如Life Technologies公司的LipoCRISPRMax)混合，形成將RNP-脂質(zhì)體復(fù)合物，與細(xì)胞在培養(yǎng)基中共同孵育。實(shí)驗(yàn)證明，具有多個(gè)核定位序列的SpyCas9蛋比傳統(tǒng)SpyCas9具有更高的基因編輯效率。

1實(shí)驗(yàn)方法

1)EGFP報(bào)告細(xì)胞的構(gòu)建。從金唯智(南京)采用常規(guī)的基因合成方法合成含有CRISPR/Cas9識(shí)別位點(diǎn)的EGFP報(bào)告基因(SEQ ID NO：1)，并亞克隆進(jìn)入pcDNA 5/FRT載體(賽默飛，V601020)。具體步驟如下：1.酶切：50微升反應(yīng)體系：10微克含有CRISPR/Cas9識(shí)別位點(diǎn)的EGFP報(bào)告基因(金唯智提供；SEQ IDNO：1)、1微升NheI限制性內(nèi)切酶(NEB，R0131S)、1微升Xho I限制性內(nèi)切酶(NEB，R0146S)、1×NEB buffer 4(NEB，B7004S)，加水補(bǔ)足50微升。在37℃處理4小時(shí)，得到EGFP基因(SEQ ID：14～SEQ ID：24)。對(duì)pcDNA 5/FRT載體同樣處理，得到pcDNA 5/FRT酶切載體。用膠回收試劑盒(Qiagen，28704)分別對(duì)EGFP基因和pcDNA 5/FRT目的片段進(jìn)行回收。2.連接。在10微升反應(yīng)體系：50微克純化的pcDNA 5/FRT酶切載體、25微克純化的EGFP基因(SEQ ID：14～SEQ ID：24)、1微升T4DNA連接酶(NEB，M0202S)，1×T4DNA連接酶緩沖液(NEB，B0202S)，加水補(bǔ)足10微升。在16℃反應(yīng)過(guò)夜，得到連接產(chǎn)物。3.轉(zhuǎn)化。將5微升連接產(chǎn)物按照說(shuō)明轉(zhuǎn)化進(jìn)入Trans10化學(xué)轉(zhuǎn)化感受態(tài)細(xì)胞(全式金，CD-101-01)，將轉(zhuǎn)化克隆送往金唯智按照常規(guī)方法提取質(zhì)粒并進(jìn)行測(cè)序驗(yàn)證，獲得重組pcDNA 5/FRT-EGFP質(zhì)粒。利用Flp-In試劑盒(賽默飛，K6010-01)，將pcDNA 5/FRT-EGFP質(zhì)粒按照試劑盒操作說(shuō)明通過(guò)重組方式插入試劑盒中HEK293Flp-In細(xì)胞的基因組，獲得HEK293EGFP報(bào)告細(xì)胞。

2)SpyCas9-sgRNA RNP的準(zhǔn)備。如實(shí)施例1中所述。

3)將1微升SpyCas9-sgRNARNP與1.5微升lipofectamine CRISPRMAX轉(zhuǎn)染試劑(賽默飛，CMAX00015)按照試劑操作說(shuō)明混合，然后加入2×10⁵個(gè)HEK293EGFP報(bào)告細(xì)胞中。在37℃、5％二氧化碳、飽和濕度的恒溫培養(yǎng)箱中培養(yǎng)48小時(shí)后，用細(xì)胞流式儀進(jìn)行檢測(cè)。

2結(jié)果分析

通過(guò)比較發(fā)現(xiàn)，除個(gè)別樣本外(N3/C0和N4/C0)，具有多個(gè)核定位序列的SpyCas9蛋白均呈現(xiàn)出顯著的活性提升(附圖3)。

實(shí)施例3顯微注射方式的細(xì)胞運(yùn)輸

本實(shí)施例將純化的具有多個(gè)核定位序列的SpyCas9蛋白，與體外轉(zhuǎn)錄的sgRNA共同孵育，形成核酸蛋白復(fù)合物(RNP)，將此RNP注射進(jìn)受精卵。具有多個(gè)核定位序列的SpyCas9蛋白比傳統(tǒng)SpyCas9具有更高的基因編輯效率。

1實(shí)驗(yàn)方法

1)小鼠胚胎的獲取。小鼠受精卵細(xì)胞購(gòu)自南方模式動(dòng)物(上海)公司。

2)SpyCas9-sgRNA RNP的準(zhǔn)備。如實(shí)施例1中所述。

3)將SpyCas9-sgRNA RNP用胚胎注射緩沖液(10mM Tris-HCl(Sigma-Aldrich，000000010708976001)，pH 7.4，0.25mM EDTA(Sigma-Aldrich，03620-50G))稀釋到50微克/微升濃度，通過(guò)顯微注射將10pL的稀釋后RNP注射進(jìn)入受精卵。受精卵在含有氨基酸的KSOM培養(yǎng)基(Millipore，MR-121-D)中培養(yǎng)在37℃及5％二氧化碳的恒溫培養(yǎng)箱中。第3.5天8細(xì)胞期，將每個(gè)發(fā)育好的囊胚用6微升的QuickExtract(Epicentre，QEB0905T)緩沖液裂解后，用PTEN基因的引物PTEN-F(SEQ ID：8)和PTEN-R(SEQ ID：9)進(jìn)行PCR擴(kuò)增(20微升的PCR溶液：1微升QuickExtract裂解液、1×Taq Master Mix(NEB，M0270L)，0.2μM的引物PTEN-F(SEQ ID：8)和PTEN-R(SEQ ID：8)，加水補(bǔ)足20微升；PCR條件為：95℃10分鐘，40個(gè)循環(huán)的95℃30秒、55℃30秒以及72℃1分鐘，最后加上72℃10分鐘)，測(cè)序(金唯智，南京)驗(yàn)證基因編輯效率。

2結(jié)果分析

通過(guò)比較發(fā)現(xiàn)(附圖4)，原始未經(jīng)改造(N0/C1)的SpyCas9在12個(gè)顯微注射并成功發(fā)育成8細(xì)胞期的胚胎中只檢測(cè)到了3個(gè)含有突變(25％)；而經(jīng)過(guò)改造的SpyCas9(N0/C4)在7個(gè)顯微注射并成功發(fā)育成8細(xì)胞期的胚胎中檢測(cè)到了5個(gè)含有突變(71.4％)。

SEQUENCE LISTING

<110>上海科技大學(xué)

<120> 具有多個(gè)核定位序列的SpyCas9蛋白及其應(yīng)用

<160> 24

<170> PatentIn version 3.3

<210> 1

<211> 814

<212> DNA

<213> EGFP reporter gene

<220>

<221> Cas9識(shí)別位點(diǎn)

<222> (486)..(530)

<400> 1

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gtcgccggtg 480

tcttccagaa ttgatactga ctgtatggat tcagtcagta tcaattctgg aagattgtag 540

ggataacagg gtaatcccgc cggtgcagaa gaacggcatc aaggtgaact tcaagatccg 600

ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 660

cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag 720

caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 780

gatcactctc ggcatggacg agctgtacaa gtaa 814

<210> 2

<211> 60

<212> DNA

<213> sgRNACCR5-F

<400> 2

taatacgact cactataggg tgacatcaat tattatacat gttttagagc tagaaatagc 60

<210> 3

<211> 60

<212> DNA

<213> sgRNAEGFP-F

<400> 3

taatacgact cactataggg cagaattgat actgactgta gttttagagc tagaaatagc 60

<210> 4

<211> 60

<212> DNA

<213> sgRNAPTEN-F

<400> 4

taatacgact cactataggg acaatattga tgatgtagta gttttagagc tagaaatagc 60

<210> 5

<211> 18

<212> DNA

<213> sgRNA-R

<400> 5

agcaccgact cggtgcca 18

<210> 6

<211> 21

<212> DNA

<213> CCR5-F

<400> 6

gagccaagct ctccatctag t 21

<210> 7

<211> 20

<212> DNA

<213> CCR5-R

<400> 7

gccctgtcaa gagttgacac 20

<210> 8

<211> 20

<212> DNA

<213> PTEN-F

<400> 8

gagcctcagt cgcgtattct 20

<210> 9

<211> 20

<212> DNA

<213> PTEN-R

<400> 9

caagccagag aataacctgg 20

<210> 10

<211> 1392

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> HA tag

<222> (1373)..(1381)

<220>

<221> His tag

<222> (1387)..(1392)

<400> 10

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Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1205 1210 1215

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

Gly Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu Gly

1370 1375 1380

Gly Gly Ser His His His His His His

1385 1390

<210> 11

<211> 1408

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> NLS sequence N-terminal

<222> (5)..(11)

<220>

<221> HA tag

<222> (1389)..(1396)

<220>

<221> His tag

<222> (1403)..(1408)

<400> 11

Met Gly Ser Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Thr Ser

1 5 10 15

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

20 25 30

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

35 40 45

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

50 55 60

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

65 70 75 80

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

85 90 95

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

100 105 110

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

115 120 125

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

130 135 140

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

145 150 155 160

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

165 170 175

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

180 185 190

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

195 200 205

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

210 215 220

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

225 230 235 240

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

245 250 255

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

260 265 270

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

275 280 285

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

290 295 300

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

305 310 315 320

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

325 330 335

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

340 345 350

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

355 360 365

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

370 375 380

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

385 390 395 400

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

405 410 415

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

420 425 430

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

435 440 445

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

450 455 460

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

465 470 475 480

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

485 490 495

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

500 505 510

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

515 520 525

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

530 535 540

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

545 550 555 560

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

565 570 575

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

580 585 590

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

595 600 605

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

610 615 620

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

625 630 635 640

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

645 650 655

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

660 665 670

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

675 680 685

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

690 695 700

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

705 710 715 720

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

725 730 735

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

740 745 750

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

755 760 765

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

770 775 780

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

785 790 795 800

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

805 810 815

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

820 825 830

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

835 840 845

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

850 855 860

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

865 870 875 880

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

885 890 895

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

900 905 910

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

915 920 925

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

930 935 940

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

945 950 955 960

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

965 970 975

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

980 985 990

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

995 1000 1005

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu

1010 1015 1020

Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile

1025 1030 1035

Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe

1040 1045 1050

Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu

1055 1060 1065

Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly

1070 1075 1080

Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr

1085 1090 1095

Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys

1100 1105 1110

Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro

1115 1120 1125

Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp

1130 1135 1140

Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser

1145 1150 1155

Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu

1160 1165 1170

Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser

1175 1180 1185

Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr

1190 1195 1200

Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser

1205 1210 1215

Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala

1220 1225 1230

Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1235 1240 1245

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly

1250 1255 1260

Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His

1265 1270 1275

Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser

1280 1285 1290

Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser

1295 1300 1305

Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu

1310 1315 1320

Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala

1325 1330 1335

Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr

1340 1345 1350

Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile

1355 1360 1365

Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly

1370 1375 1380

Asp Gly Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu

1385 1390 1395

Gly Gly Gly Ser His His His His His His

1400 1405

<210> 12

<211> 1405

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> HA tag

<222> (1372)..(1380)

<220>

<221> NLS sequence C-terminal

<222> (1390)..(1396)

<220>

<221> His tag

<222> (1400)..(1405)

<400> 12

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1010 1015 1020

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1055 1060 1065

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1205 1210 1215

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

Gly Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu Gly

1370 1375 1380

Gly Gly Arg Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly

1385 1390 1395

Ser His His His His His His

1400 1405

<210> 13

<211> 1421

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> NLS sequence N-terminal

<222> (5)..(11)

<220>

<221> HA tag

<222> (1388)..(1396)

<220>

<221> NLS sequence C-terminal

<222> (1406)..(1412)

<220>

<221> His tag

<222> (1416)..(1421)

<400> 13

Met Gly Ser Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Thr Ser

1 5 10 15

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

20 25 30

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

35 40 45

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

50 55 60

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

65 70 75 80

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

85 90 95

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

100 105 110

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

115 120 125

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

130 135 140

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

145 150 155 160

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

165 170 175

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

180 185 190

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

195 200 205

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

210 215 220

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

225 230 235 240

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

245 250 255

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

260 265 270

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

275 280 285

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

290 295 300

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

305 310 315 320

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

325 330 335

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

340 345 350

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

355 360 365

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

370 375 380

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

385 390 395 400

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

405 410 415

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

420 425 430

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

435 440 445

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

450 455 460

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

465 470 475 480

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

485 490 495

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

500 505 510

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

515 520 525

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

530 535 540

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

545 550 555 560

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

565 570 575

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

580 585 590

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

595 600 605

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

610 615 620

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

625 630 635 640

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

645 650 655

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

660 665 670

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

675 680 685

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

690 695 700

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

705 710 715 720

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

725 730 735

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

740 745 750

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

755 760 765

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

770 775 780

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

785 790 795 800

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

805 810 815

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

820 825 830

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

835 840 845

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

850 855 860

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

865 870 875 880

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

885 890 895

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

900 905 910

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

915 920 925

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

930 935 940

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

945 950 955 960

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

965 970 975

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

980 985 990

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

995 1000 1005

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu

1010 1015 1020

Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile

1025 1030 1035

Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe

1040 1045 1050

Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu

1055 1060 1065

Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly

1070 1075 1080

Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr

1085 1090 1095

Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys

1100 1105 1110

Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro

1115 1120 1125

Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp

1130 1135 1140

Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser

1145 1150 1155

Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu

1160 1165 1170

Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser

1175 1180 1185

Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr

1190 1195 1200

Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser

1205 1210 1215

Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala

1220 1225 1230

Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1235 1240 1245

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly

1250 1255 1260

Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His

1265 1270 1275

Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser

1280 1285 1290

Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser

1295 1300 1305

Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu

1310 1315 1320

Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala

1325 1330 1335

Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr

1340 1345 1350

Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile

1355 1360 1365

Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly

1370 1375 1380

Asp Gly Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu

1385 1390 1395

Gly Gly Gly Arg Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly

1400 1405 1410

Gly Ser His His His His His His

1415 1420

<210> 14

<211> 1415

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> HA tag

<222> (1372)..(1379)

<220>

<221> NLS sequence C-terminal

<222> (1387)..(1406)

<220>

<221> His tag

<222> (1410)..(1415)

<400> 14

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1010 1015 1020

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1055 1060 1065

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1205 1210 1215

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

Gly Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu Gly

1370 1375 1380

Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Arg Gly Gly

1385 1390 1395

Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser His His His His

1400 1405 1410

His His

1415

<210> 15

<211> 1425

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> HA tag

<222> (1372)..(1380)

<220>

<221> NLS sequence C-terminal

<222> (1387)..(1416)

<220>

<221> His tag

<222> (1420)..(1425)

<400> 15

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1010 1015 1020

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1055 1060 1065

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1205 1210 1215

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

Gly Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu Gly

1370 1375 1380

Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Pro Lys

1385 1390 1395

Lys Lys Arg Lys Val Gly Gly Arg Gly Gly Ser Pro Lys Lys Lys

1400 1405 1410

Arg Lys Val Gly Gly Ser His His His His His His

1415 1420 1425

<210> 16

<211> 1435

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> HA tag

<222> (1372)..(1380)

<220>

<221> NLS sequence C-terminal

<222> (1387)..(1426)

<220>

<221> His tag

<222> (1430)..(1435)

<400> 16

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1010 1015 1020

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1025 1030 1035

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1040 1045 1050

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1055 1060 1065

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1085 1090 1095

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1100 1105 1110

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1145 1150 1155

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1160 1165 1170

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1175 1180 1185

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1205 1210 1215

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1250 1255 1260

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1265 1270 1275

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1295 1300 1305

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1340 1345 1350

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

Gly Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu Gly

1370 1375 1380

Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Pro Lys

1385 1390 1395

Lys Lys Arg Lys Val Gly Gly Ser Pro Lys Lys Lys Arg Lys Val

1400 1405 1410

Gly Gly Arg Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly

1415 1420 1425

Ser His His His His His His

1430 1435

<210> 17

<211> 1431

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> NLS sequence N-terminal

<222> (5)..(11)

<220>

<221> HA tag

<222> (1388)..(1396)

<220>

<221> NLS sequence C-terminal

<222> (1403)..(1409)

<220>

<221> His tag

<222> (1426)..(1431)

<400> 17

Met Gly Ser Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Thr Ser

1 5 10 15

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

20 25 30

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

35 40 45

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

50 55 60

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

65 70 75 80

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

85 90 95

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

100 105 110

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

115 120 125

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

130 135 140

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

145 150 155 160

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

165 170 175

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

180 185 190

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

195 200 205

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

210 215 220

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

225 230 235 240

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

245 250 255

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

260 265 270

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

275 280 285

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

290 295 300

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

305 310 315 320

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

325 330 335

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

340 345 350

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

355 360 365

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

370 375 380

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

385 390 395 400

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

405 410 415

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

420 425 430

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

435 440 445

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

450 455 460

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

465 470 475 480

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

485 490 495

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

500 505 510

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

515 520 525

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

530 535 540

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

545 550 555 560

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

565 570 575

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

580 585 590

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

595 600 605

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

610 615 620

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

625 630 635 640

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

645 650 655

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

660 665 670

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

675 680 685

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

690 695 700

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

705 710 715 720

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

725 730 735

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

740 745 750

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

755 760 765

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

770 775 780

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

785 790 795 800

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

805 810 815

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

820 825 830

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

835 840 845

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

850 855 860

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

865 870 875 880

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

885 890 895

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

900 905 910

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

915 920 925

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

930 935 940

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

945 950 955 960

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

965 970 975

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

980 985 990

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

995 1000 1005

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu

1010 1015 1020

Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile

1025 1030 1035

Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe

1040 1045 1050

Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu

1055 1060 1065

Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly

1070 1075 1080

Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr

1085 1090 1095

Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys

1100 1105 1110

Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro

1115 1120 1125

Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp

1130 1135 1140

Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser

1145 1150 1155

Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu

1160 1165 1170

Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser

1175 1180 1185

Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr

1190 1195 1200

Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser

1205 1210 1215

Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala

1220 1225 1230

Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1235 1240 1245

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly

1250 1255 1260

Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His

1265 1270 1275

Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser

1280 1285 1290

Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser

1295 1300 1305

Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu

1310 1315 1320

Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala

1325 1330 1335

Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr

1340 1345 1350

Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile

1355 1360 1365

Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly

1370 1375 1380

Asp Gly Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu

1385 1390 1395

Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Arg Gly

1400 1405 1410

Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser His His His

1415 1420 1425

His His His

1430

<210> 18

<211> 1441

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> NLS sequence N-terminal

<222> (5)..(11)

<220>

<221> HA tag

<222> (1388)..(1396)

<220>

<221> NLS sequence C-terminal

<222> (1403)..(1432)

<220>

<221> His tag

<222> (1436)..(1441)

<400> 18

Met Gly Ser Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Thr Ser

1 5 10 15

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

20 25 30

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

35 40 45

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

50 55 60

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

65 70 75 80

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

85 90 95

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

100 105 110

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

115 120 125

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

130 135 140

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

145 150 155 160

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

165 170 175

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

180 185 190

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

195 200 205

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

210 215 220

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

225 230 235 240

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

245 250 255

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

260 265 270

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

275 280 285

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

290 295 300

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

305 310 315 320

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

325 330 335

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

340 345 350

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

355 360 365

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

370 375 380

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

385 390 395 400

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

405 410 415

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

420 425 430

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

435 440 445

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

450 455 460

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

465 470 475 480

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

485 490 495

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

500 505 510

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

515 520 525

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

530 535 540

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

545 550 555 560

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

565 570 575

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

580 585 590

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

595 600 605

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

610 615 620

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

625 630 635 640

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

645 650 655

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

660 665 670

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

675 680 685

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

690 695 700

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

705 710 715 720

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

725 730 735

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

740 745 750

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

755 760 765

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

770 775 780

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

785 790 795 800

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

805 810 815

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

820 825 830

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

835 840 845

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

850 855 860

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

865 870 875 880

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

885 890 895

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

900 905 910

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

915 920 925

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

930 935 940

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

945 950 955 960

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

965 970 975

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

980 985 990

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

995 1000 1005

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu

1010 1015 1020

Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile

1025 1030 1035

Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe

1040 1045 1050

Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu

1055 1060 1065

Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly

1070 1075 1080

Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr

1085 1090 1095

Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys

1100 1105 1110

Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro

1115 1120 1125

Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp

1130 1135 1140

Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser

1145 1150 1155

Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu

1160 1165 1170

Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser

1175 1180 1185

Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr

1190 1195 1200

Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser

1205 1210 1215

Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala

1220 1225 1230

Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1235 1240 1245

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly

1250 1255 1260

Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His

1265 1270 1275

Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser

1280 1285 1290

Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser

1295 1300 1305

Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu

1310 1315 1320

Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala

1325 1330 1335

Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr

1340 1345 1350

Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile

1355 1360 1365

Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly

1370 1375 1380

Asp Gly Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu

1385 1390 1395

Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Pro

1400 1405 1410

Lys Lys Lys Arg Lys Val Gly Gly Arg Gly Gly Ser Pro Lys Lys

1415 1420 1425

Lys Arg Lys Val Gly Gly Ser His His His His His His

1430 1435 1440

<210> 19

<211> 1418

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> NLS sequence N-terminal

<222> (5)..(23)

<220>

<221> HA tag

<222> (1398)..(1406)

<220>

<221> His tag

<222> (1413)..(1418)

<400> 19

Met Gly Ser Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Thr Ser

1 5 10 15

Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Met Asp Lys Lys Tyr Ser

20 25 30

Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr

35 40 45

Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr

50 55 60

Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp

65 70 75 80

Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg

85 90 95

Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe

100 105 110

Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu

115 120 125

Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile

130 135 140

Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr

145 150 155 160

Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp

165 170 175

Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly

180 185 190

His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp

195 200 205

Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu

210 215 220

Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala

225 230 235 240

Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro

245 250 255

Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu

260 265 270

Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala

275 280 285

Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu

290 295 300

Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys

305 310 315 320

Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr

325 330 335

Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp

340 345 350

Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln

355 360 365

Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly

370 375 380

Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys

385 390 395 400

Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu

405 410 415

Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp

420 425 430

Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile

435 440 445

Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu

450 455 460

Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro

465 470 475 480

Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu

485 490 495

Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala

500 505 510

Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu

515 520 525

Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe

530 535 540

Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met

545 550 555 560

Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp

565 570 575

Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu

580 585 590

Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly

595 600 605

Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu

610 615 620

Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp

625 630 635 640

Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu

645 650 655

Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys

660 665 670

Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu

675 680 685

Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr

690 695 700

Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met

705 710 715 720

Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys

725 730 735

Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn

740 745 750

Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys

755 760 765

Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn

770 775 780

Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln

785 790 795 800

Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu

805 810 815

Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu

820 825 830

Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met

835 840 845

Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val

850 855 860

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn

865 870 875 880

Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val

885 890 895

Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu

900 905 910

Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys

915 920 925

Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys

930 935 940

Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile

945 950 955 960

Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile

965 970 975

Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe

980 985 990

Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His

995 1000 1005

His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu

1010 1015 1020

Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp

1025 1030 1035

Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln

1040 1045 1050

Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile

1055 1060 1065

Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile

1070 1075 1080

Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1085 1090 1095

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu

1100 1105 1110

Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr

1115 1120 1125

Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp

1130 1135 1140

Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly

1145 1150 1155

Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala

1160 1165 1170

Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu

1175 1180 1185

Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn

1190 1195 1200

Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys

1205 1210 1215

Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu

1220 1225 1230

Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys

1235 1240 1245

Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr

1250 1255 1260

Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn

1265 1270 1275

Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp

1280 1285 1290

Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu

1295 1300 1305

Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His

1310 1315 1320

Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1325 1330 1335

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe

1340 1345 1350

Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val

1355 1360 1365

Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu

1370 1375 1380

Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Gly Gly Ser Tyr

1385 1390 1395

Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu Gly Gly Gly Ser His

1400 1405 1410

His His His His His

1415

<210> 20

<211> 1431

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> NLS sequence N-terminal

<222> (5)..(23)

<220>

<221> HA tag

<222> (1398)..(1406)

<220>

<221> NLS sequence C-terminal

<222> (1416)..(1422)

<220>

<221> His tag

<222> (1426)..(1431)

<400> 20

Met Gly Ser Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Thr Ser

1 5 10 15

Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Met Asp Lys Lys Tyr Ser

20 25 30

Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr

35 40 45

Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr

50 55 60

Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp

65 70 75 80

Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg

85 90 95

Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe

100 105 110

Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu

115 120 125

Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile

130 135 140

Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr

145 150 155 160

Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp

165 170 175

Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly

180 185 190

His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp

195 200 205

Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu

210 215 220

Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala

225 230 235 240

Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro

245 250 255

Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu

260 265 270

Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala

275 280 285

Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu

290 295 300

Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys

305 310 315 320

Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr

325 330 335

Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp

340 345 350

Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln

355 360 365

Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly

370 375 380

Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys

385 390 395 400

Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu

405 410 415

Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp

420 425 430

Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile

435 440 445

Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu

450 455 460

Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro

465 470 475 480

Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu

485 490 495

Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala

500 505 510

Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu

515 520 525

Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe

530 535 540

Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met

545 550 555 560

Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp

565 570 575

Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu

580 585 590

Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly

595 600 605

Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu

610 615 620

Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp

625 630 635 640

Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu

645 650 655

Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys

660 665 670

Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu

675 680 685

Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr

690 695 700

Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met

705 710 715 720

Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys

725 730 735

Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn

740 745 750

Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys

755 760 765

Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn

770 775 780

Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln

785 790 795 800

Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu

805 810 815

Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu

820 825 830

Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met

835 840 845

Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val

850 855 860

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn

865 870 875 880

Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val

885 890 895

Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu

900 905 910

Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys

915 920 925

Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys

930 935 940

Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile

945 950 955 960

Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile

965 970 975

Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe

980 985 990

Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His

995 1000 1005

His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu

1010 1015 1020

Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp

1025 1030 1035

Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln

1040 1045 1050

Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile

1055 1060 1065

Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile

1070 1075 1080

Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1085 1090 1095

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu

1100 1105 1110

Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr

1115 1120 1125

Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp

1130 1135 1140

Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly

1145 1150 1155

Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala

1160 1165 1170

Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu

1175 1180 1185

Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn

1190 1195 1200

Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys

1205 1210 1215

Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu

1220 1225 1230

Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys

1235 1240 1245

Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr

1250 1255 1260

Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn

1265 1270 1275

Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp

1280 1285 1290

Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu

1295 1300 1305

Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His

1310 1315 1320

Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1325 1330 1335

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe

1340 1345 1350

Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val

1355 1360 1365

Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu

1370 1375 1380

Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Gly Gly Ser Tyr

1385 1390 1395

Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu Gly Gly Gly Arg Gly

1400 1405 1410

Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser His His His

1415 1420 1425

His His His

1430

<210> 21

<211> 1440

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> NLS sequence N-terminal

<222> (5)..(23)

<220>

<221> HA tag

<222> (1398)..(1406)

<220>

<221> NLS sequence C-terminal

<222> (1412)..(1431)

<220>

<221> His tag

<222> (1435)..(1440)

<400> 21

Met Gly Ser Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Thr Ser

1 5 10 15

Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Met Asp Lys Lys Tyr Ser

20 25 30

Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr

35 40 45

Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr

50 55 60

Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp

65 70 75 80

Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg

85 90 95

Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe

100 105 110

Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu

115 120 125

Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile

130 135 140

Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr

145 150 155 160

Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp

165 170 175

Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly

180 185 190

His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp

195 200 205

Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu

210 215 220

Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala

225 230 235 240

Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro

245 250 255

Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu

260 265 270

Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala

275 280 285

Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu

290 295 300

Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys

305 310 315 320

Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr

325 330 335

Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp

340 345 350

Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln

355 360 365

Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly

370 375 380

Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys

385 390 395 400

Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu

405 410 415

Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp

420 425 430

Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile

435 440 445

Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu

450 455 460

Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro

465 470 475 480

Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu

485 490 495

Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala

500 505 510

Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu

515 520 525

Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe

530 535 540

Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met

545 550 555 560

Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp

565 570 575

Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu

580 585 590

Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly

595 600 605

Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu

610 615 620

Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp

625 630 635 640

Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu

645 650 655

Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys

660 665 670

Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu

675 680 685

Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr

690 695 700

Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met

705 710 715 720

Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys

725 730 735

Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn

740 745 750

Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys

755 760 765

Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn

770 775 780

Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln

785 790 795 800

Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu

805 810 815

Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu

820 825 830

Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met

835 840 845

Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val

850 855 860

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn

865 870 875 880

Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val

885 890 895

Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu

900 905 910

Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys

915 920 925

Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys

930 935 940

Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile

945 950 955 960

Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile

965 970 975

Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe

980 985 990

Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His

995 1000 1005

His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu

1010 1015 1020

Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp

1025 1030 1035

Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln

1040 1045 1050

Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile

1055 1060 1065

Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile

1070 1075 1080

Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1085 1090 1095

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu

1100 1105 1110

Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr

1115 1120 1125

Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp

1130 1135 1140

Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly

1145 1150 1155

Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala

1160 1165 1170

Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu

1175 1180 1185

Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn

1190 1195 1200

Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys

1205 1210 1215

Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu

1220 1225 1230

Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys

1235 1240 1245

Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr

1250 1255 1260

Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn

1265 1270 1275

Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp

1280 1285 1290

Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu

1295 1300 1305

Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His

1310 1315 1320

Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1325 1330 1335

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe

1340 1345 1350

Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val

1355 1360 1365

Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu

1370 1375 1380

Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Gly Gly Ser Tyr

1385 1390 1395

Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu Gly Gly Ser Pro Lys

1400 1405 1410

Lys Lys Arg Lys Val Gly Gly Arg Gly Gly Ser Pro Lys Lys Lys

1415 1420 1425

Arg Lys Val Gly Gly Ser His His His His His His

1430 1435 1440

<210> 22

<211> 1428

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> NLS sequence N-terminal

<222> (5)..(33)

<220>

<221> HA tag

<222> (1408)..(1416)

<220>

<221> His tag

<222> (1423)..(1428)

<400> 22

Met Gly Ser Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Thr Ser

1 5 10 15

Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Pro Lys Lys Lys Arg Lys

20 25 30

Val Gly Gly Ser Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly

35 40 45

Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro

50 55 60

Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys

65 70 75 80

Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu

85 90 95

Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys

100 105 110

Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys

115 120 125

Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu

130 135 140

Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp

145 150 155 160

Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys

165 170 175

Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu

180 185 190

Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly

195 200 205

Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu

210 215 220

Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser

225 230 235 240

Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg

245 250 255

Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly

260 265 270

Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe

275 280 285

Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys

290 295 300

Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp

305 310 315 320

Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile

325 330 335

Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro

340 345 350

Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu

355 360 365

Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys

370 375 380

Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp

385 390 395 400

Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu

405 410 415

Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu

420 425 430

Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His

435 440 445

Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp

450 455 460

Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu

465 470 475 480

Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser

485 490 495

Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp

500 505 510

Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile

515 520 525

Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu

530 535 540

Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu

545 550 555 560

Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu

565 570 575

Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn

580 585 590

Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile

595 600 605

Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn

610 615 620

Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys

625 630 635 640

Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val

645 650 655

Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu

660 665 670

Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys

675 680 685

Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn

690 695 700

Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys

705 710 715 720

Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp

725 730 735

Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln

740 745 750

Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala

755 760 765

Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val

770 775 780

Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala

785 790 795 800

Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg

805 810 815

Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu

820 825 830

Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr

835 840 845

Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu

850 855 860

Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln

865 870 875 880

Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser

885 890 895

Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val

900 905 910

Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile

915 920 925

Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu

930 935 940

Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr

945 950 955 960

Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn

965 970 975

Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile

980 985 990

Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe

995 1000 1005

Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala

1010 1015 1020

Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro

1025 1030 1035

Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp

1040 1045 1050

Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala

1055 1060 1065

Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys

1070 1075 1080

Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu

1085 1090 1095

Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly

1100 1105 1110

Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val

1115 1120 1125

Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys

1130 1135 1140

Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg

1145 1150 1155

Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro

1160 1165 1170

Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1175 1180 1185

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr

1190 1195 1200

Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu

1205 1210 1215

Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys

1220 1225 1230

Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg

1235 1240 1245

Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala

1250 1255 1260

Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr

1265 1270 1275

Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu

1280 1285 1290

Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln

1295 1300 1305

Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu

1310 1315 1320

Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile

1325 1330 1335

Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn

1340 1345 1350

Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp

1355 1360 1365

Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu

1370 1375 1380

Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu

1385 1390 1395

Ser Gln Leu Gly Gly Asp Gly Gly Ser Tyr Pro Tyr Asp Val Pro

1400 1405 1410

Asp Tyr Ala Leu Glu Gly Gly Gly Ser His His His His His His

1415 1420 1425

<210> 23

<211> 1441

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> NLS sequence N-terminal

<222> (5)..(33)

<220>

<221> HA tag

<222> (1408)..(1416)

<220>

<221> NLS sequence C-terminal

<222> (1426)..(1432)

<220>

<221> His tag

<222> (1436)..(1441)

<400> 23

Met Gly Ser Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Thr Ser

1 5 10 15

Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Pro Lys Lys Lys Arg Lys

20 25 30

Val Gly Gly Ser Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly

35 40 45

Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro

50 55 60

Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys

65 70 75 80

Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu

85 90 95

Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys

100 105 110

Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys

115 120 125

Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu

130 135 140

Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp

145 150 155 160

Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys

165 170 175

Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu

180 185 190

Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly

195 200 205

Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu

210 215 220

Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser

225 230 235 240

Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg

245 250 255

Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly

260 265 270

Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe

275 280 285

Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys

290 295 300

Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp

305 310 315 320

Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile

325 330 335

Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro

340 345 350

Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu

355 360 365

Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys

370 375 380

Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp

385 390 395 400

Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu

405 410 415

Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu

420 425 430

Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His

435 440 445

Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp

450 455 460

Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu

465 470 475 480

Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser

485 490 495

Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp

500 505 510

Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile

515 520 525

Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu

530 535 540

Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu

545 550 555 560

Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu

565 570 575

Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn

580 585 590

Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile

595 600 605

Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn

610 615 620

Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys

625 630 635 640

Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val

645 650 655

Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu

660 665 670

Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys

675 680 685

Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn

690 695 700

Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys

705 710 715 720

Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp

725 730 735

Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln

740 745 750

Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala

755 760 765

Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val

770 775 780

Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala

785 790 795 800

Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg

805 810 815

Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu

820 825 830

Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr

835 840 845

Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu

850 855 860

Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln

865 870 875 880

Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser

885 890 895

Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val

900 905 910

Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile

915 920 925

Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu

930 935 940

Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr

945 950 955 960

Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn

965 970 975

Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile

980 985 990

Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe

995 1000 1005

Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala

1010 1015 1020

Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro

1025 1030 1035

Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp

1040 1045 1050

Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala

1055 1060 1065

Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys

1070 1075 1080

Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu

1085 1090 1095

Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly

1100 1105 1110

Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val

1115 1120 1125

Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys

1130 1135 1140

Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg

1145 1150 1155

Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro

1160 1165 1170

Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1175 1180 1185

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr

1190 1195 1200

Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu

1205 1210 1215

Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys

1220 1225 1230

Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg

1235 1240 1245

Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala

1250 1255 1260

Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr

1265 1270 1275

Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu

1280 1285 1290

Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln

1295 1300 1305

Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu

1310 1315 1320

Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile

1325 1330 1335

Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn

1340 1345 1350

Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp

1355 1360 1365

Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu

1370 1375 1380

Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu

1385 1390 1395

Ser Gln Leu Gly Gly Asp Gly Gly Ser Tyr Pro Tyr Asp Val Pro

1400 1405 1410

Asp Tyr Ala Leu Glu Gly Gly Gly Arg Gly Gly Ser Pro Lys Lys

1415 1420 1425

Lys Arg Lys Val Gly Gly Ser His His His His His His

1430 1435 1440

<210> 24

<211> 1438

<212> PRT

<213> Streptococcus pyogenes

<220>

<221> NLS sequence N-terminal

<222> (5)..(43)

<220>

<221> HA tag

<222> (1418)..(1426)

<220>

<221> His tag

<222> (1433)..(1437)

<400> 24

Met Gly Ser Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Thr Ser

1 5 10 15

Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Pro Lys Lys Lys Arg Lys

20 25 30

Val Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Met Asp

35 40 45

Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp

50 55 60

Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val

65 70 75 80

Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala

85 90 95

Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg

100 105 110

Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu

115 120 125

Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe

130 135 140

His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu

145 150 155 160

Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu

165 170 175

Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr

180 185 190

Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile

195 200 205

Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn

210 215 220

Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln

225 230 235 240

Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala

245 250 255

Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile

260 265 270

Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile

275 280 285

Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu

290 295 300

Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp

305 310 315 320

Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe

325 330 335

Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu

340 345 350

Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile

355 360 365

Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu

370 375 380

Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln

385 390 395 400

Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu

405 410 415

Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr

420 425 430

Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln

435 440 445

Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu

450 455 460

Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys

465 470 475 480

Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr

485 490 495

Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr

500 505 510

Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val

515 520 525

Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe

530 535 540

Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu

545 550 555 560

Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val

565 570 575

Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys

580 585 590

Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys

595 600 605

Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val

610 615 620

Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr

625 630 635 640

His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu

645 650 655

Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe

660 665 670

Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu

675 680 685

Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly

690 695 700

Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln

705 710 715 720

Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn

725 730 735

Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu

740 745 750

Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu

755 760 765

His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu

770 775 780

Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His

785 790 795 800

Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr

805 810 815

Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu

820 825 830

Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu

835 840 845

Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn

850 855 860

Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser

865 870 875 880

Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp

885 890 895

Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys

900 905 910

Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr

915 920 925

Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp

930 935 940

Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala

945 950 955 960

Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His

965 970 975

Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn

980 985 990

Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu

995 1000 1005

Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu

1010 1015 1020

Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

1025 1030 1035

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu

1040 1045 1050

Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile

1055 1060 1065

Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe

1070 1075 1080

Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu

1085 1090 1095

Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly

1100 1105 1110

Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr

1115 1120 1125

Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys

1130 1135 1140

Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro

1145 1150 1155

Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp

1160 1165 1170

Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser

1175 1180 1185

Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu

1190 1195 1200

Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser

1205 1210 1215

Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr

1220 1225 1230

Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser

1235 1240 1245

Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala

1250 1255 1260

Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1265 1270 1275

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly

1280 1285 1290

Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His

1295 1300 1305

Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser

1310 1315 1320

Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser

1325 1330 1335

Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu

1340 1345 1350

Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala

1355 1360 1365

Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr

1370 1375 1380

Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile

1385 1390 1395

Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly

1400 1405 1410

Asp Gly Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Leu Glu

1415 1420 1425

Gly Gly Gly Ser His His His His His His

1430 1435