CN101310023A - 用于生产重组蛋白质的翻译融合配偶体文库和从中筛选的翻译融合配偶体 - Google Patents
用于生产重组蛋白质的翻译融合配偶体文库和从中筛选的翻译融合配偶体 Download PDFInfo
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Abstract
本发明涉及快速筛选适当的翻译融合配偶体(TFPs)的技术,所述的翻译融合配偶体能够诱导重组蛋白质特别是使用常规制备方法难以生产的蛋白质的分泌生产。
Description
发明背景
发明领域
本发明的领域是重组蛋白质的表达。特别是,本发明涉及快速筛选适合的翻译融合配偶体(TFPs)的技术,其中所述的翻译融合配偶体(TFPs)能够诱导重组蛋白质,尤其是用常规重组制备方法难以生产的蛋白质的分泌表达。
相关技术
目标蛋白质的重组体表达是广泛应用的用来生产大量用于研究目的或治疗和其它商业用途的蛋白质的方法。本领域公知多种重组体表达系统,包括细菌、酵母和哺乳动物宿主细胞系统,并且许多不同蛋白质已在这些系统中成功生产。然而,还有许多蛋白质使用现有表达系统不易生产,导致很少或没有蛋白质表达和分泌。提高重组表达的蛋白质的分泌的方法,例如在宿主细胞中过表达分泌因子、使用融合蛋白、其中该融合蛋白包含与良好分泌的蛋白质融合的目标蛋白质,以及添加合成的连接序列,已在特殊的目标蛋白质上取得一定成功。但是,还没有对所有蛋白质的分泌生产都确证有效的通用技术。
在致力于鉴定分泌性蛋白质和新信号序列的努力中已开发了数个信号序列捕获系统。美国专利6,228,590描述了一种筛选哺乳动物信号序列的技术,使用能与报告蛋白融合的含有哺乳动物编码序列的核酸转化报告蛋白缺乏的酵母并检测分泌了报告蛋白的细胞。一种使用缺乏转化酶的酵母和转化酶报告蛋白的类似系统公开于欧洲专利EP0907727中。基于酵母的信号序列捕获已用于鉴定来自人DNA(Klein等人,Proc.Natl.Acad.Sci.USA 93:7108(1996);Jacobs等人,Gene 198:289(1997))、鼠DNA(Gallicioti等人,J.Membrane Biol.183:175(2001))、斑马鱼DNA(Crosier等人,Dev.Dynamics 222:637(2001))、拟南芥DNA(Goo等人,Plant Mol.Biol.41:415(1999))、马铃薯DNA(Surpili等人,Anais de Academia Brasileira de Ciencias 74:5 99(2002))和白色念珠菌DNA(Monteoliva等人,Eukaryotic Cell 1:514(2002))的分泌蛋白。已开发了使用哺乳动物宿主细胞(Gallicioti等人,J.Membrane Biol.183:175(2001))和细菌宿主细胞(Ferguson等人,CancerRes.65:8209(2000))的类似捕获系统。已用于信号序列捕获的报告蛋白包括转化酶(Klein等人,Proc.Natl.Acad.Sci.USA 93:7108(1996))、α-淀粉酶(美国专利6,228,590)、酸性磷酸酯酶(PHO5)(Surpili等人,Anais de Academia Brasileira de Ciencias 74:599(2002))和β-内酰胺酶(Ferguson等人,Cancer Res.65:8209(2000))。
证实翻译融合配偶体(TFPs)对靶蛋白的分泌有效的方法公开于WO 2005/068658中。该方法包含:(i)制备多个宿主细胞,所述的宿主细胞用包含核酸片段的文库和编码靶蛋白的核苷酸序列的载体进行转化,其中编码靶蛋白的核苷酸序列与编码报告蛋白的核苷酸序列融合,其中所述的宿主细胞缺乏报告蛋白,和(ii)从宿主细胞中鉴定出TFP文库,其中该TFP文库包含单独诱导靶蛋白分泌的核酸片段。
发明概述
本发明涉及一种快速和有效的鉴定对诱导靶蛋白分泌有效的TFPs的自动筛选方法。本发明允许宿主细胞分泌任何靶蛋白,包括使用常规重组表达系统不表达或仅低水平表达的靶蛋白。
在一个实施方案中,本发明涉及鉴定靶蛋白特异的TFP的方法,所述方法包括:
(i)用多个线性载体和编码靶蛋白的核酸序列共转化多个报告蛋白缺乏的宿主细胞来制备多个转化的宿主细胞,
其中每个所述的线性载体包含一个来自于核酸片段文库中的核酸片段和一个编码N末端氨基酸缺失的报告蛋白的核苷酸序列,和
其中所述编码靶蛋白的核酸序列在3’末端包含一个编码N末端氨基酸的核苷酸序列,其中在所述线性载体内所述报告蛋白上缺乏该N末端氨基酸;和在5’末端包含一个连接DNA;
(ii)在允许所述线性载体和所述编码靶蛋白的核酸序列体内重组的有效条件下培养所述的多个被转化的宿主细胞;
(iii)从(ii)中的多个被转化的宿主细胞中鉴定显示报告蛋白活性的细胞;和
(iv)从(iii)中鉴定的细胞中鉴定TFP;
其中所述TFP包含诱导所述靶蛋白分泌的核酸片段。
本发明的另一个实施方案涉及鉴定靶蛋白特异的TFP文库的方法,所述方法包括:
(i)用多个线性载体和一个编码靶蛋白的核酸序列共转化多个报告蛋白缺乏的宿主细胞来制备多个被转化的宿主细胞,
其中每个所述的线性载体包含一个来自于核酸片段文库中的核酸片段和一个编码N末端氨基酸缺失的报告蛋白的核苷酸序列,和
其中所述编码靶蛋白的核酸序列在3’末端包含一个编码N末端氨基酸的核苷酸序列,其中在所述线性载体内所述报告蛋白上缺乏该N末端氨基酸;和在5’末端包含一个连接DNA;
(ii)在允许所述线性载体和所述编码靶蛋白的核酸体内重组的有效条件下培养多个所述被转化的宿主细胞;
(iii)从(ii)中的多个被转化的宿主细胞中鉴定显示报告蛋白活性的细胞;和
(iv)从(iii)中鉴定的细胞中鉴定TFP文库;
其中所述TFP文库包含单独诱导所述靶蛋白分泌的核酸片段。
本发明还涉及由本发明的方法鉴定的一个TFP或TFPs文库。
本发明还包含编码TFP的核酸片段或编码TFPs的核酸片段文库。
本发明还包括一种核酸,该核酸包含编码TFP的核苷酸序列和编码靶蛋白的核苷酸序列。
本发明还涉及使用本发明的TFP制备靶蛋白的方法。
本发明还涉及一种线性载体,该线性载体包括来自核酸片段文库的核酸片段和编码N末端氨基酸缺失的报告蛋白的核苷酸序列。
本发明还包含多个用线性载体和编码本发明靶蛋白的核苷酸序列转化的报告蛋白缺乏的宿主细胞。
附图说明
本发明的上述和其它目的、特点和优点将在以下结合附图的详述中得到更好的理解。
图1显示了删除转化酶基因的方法和可选标记的敲除方法;
图2显示了转化酶活性的酶谱分析(泳道1、2和3:野生型酿酒酵母菌Y2805;和泳道4、5和6:转化酶缺失株(酿酒酵母菌Y2805Δsuc2))。
图3以照片显示了根据碳源酵母细胞的生长情况(SUC2:野生型酿酒酵母菌Y2805;和Δsuc2:转化酶缺失株(酿酒酵母菌Y2805Δsuc2))。
图4显示了转化酶基因缺失的DNA印迹的结果(泳道1和2:酿酒酵母菌Y2805(ura3SUC2);泳道3和4:酿酒酵母菌Y2805Δsuc2U(URA3Δsuc2);和泳道5和6:酿酒酵母菌Y2805Δsuc2(ura3Δsuc2))。
图5以照片分别显示了含有质粒pYGAP-SNS-SUC2、pYGAP-HSA-SUC2和pYGAP-hIL2-SUC2的酵母细胞在葡萄糖和蔗糖培养基中的生长情况。
图6显示了含有多克隆位点以在GAL10启动子和成熟转化酶基因间插入cDNA文库的质粒YGaINV的图谱。
图7显示了含有多克隆位点以在GAL10启动子和带有三个不同读码框的成熟转化酶基因间插入基因组DNA文库的质粒YGaF0INV、YGaF1INV和YGaF2INV的图谱。
图8显示了用任意引物合成cDNA文库和在TFP选择性载体YGaINV中构建cDNA文库的方法。
图9显示了在TFP选择性载体YGaF0INV、YGaF1INV和YGaF2INV中构建基因组DNA文库的方法。
图10显示了含有缺陷型SUC2的YGadV45和将TFP文库亚克隆至YGadV45的质粒图谱。
图11显示了使用TFP文库中的转化酶作为报告蛋白通过体内重组的TFP选择靶基因的方法。
图12显示了使用TFP文库中的双报告蛋白,即脂肪酶和转化酶作为报告蛋白通过体内重组的TFP选择靶基因的方法。
图13显示了含有光环形成(halo forming)转化株的三丁酸甘油酯平板:(A)直接来自转化的光环形成平板(用三丁酸甘油酯的YPSGA)、(B)在三丁酸甘油酯平板上显示不同光环大小的被选择的转化株。
图14显示了用选择的9个TFPs构建9个人IL2表达载体的方法。
图15显示了人IL2表达载体的图谱:(A)pYGT9-IL2、(B)pYGT13-IL2和(C)pYGT17-IL2。
图16显示了人IL2表达载体的图谱:(A)pYGT18-IL2,(B)pYGT19-IL2和(C)pYGT20-IL2。
图17显示了人IL2表达载体的图谱:(A)pYGT21-IL2,(B)pYGT25-IL2和(C)pYGT27-IL2。
图18显示了分泌人IL2的酵母细胞培养液上清的SDS-PAGE结果(泳道M:蛋白质大小标记;泳道1:含有pYIL-KRT1-4(WO 2005/068658)作为IL2分泌对照的酵母细胞的培养液上清;泳道2:含有pYGT9-IL2的酵母细胞的培养液上清;泳道3:含有pYGT21-IL2的酵母细胞的培养液上清;泳道4:含有pYGT13-IL2的酵母细胞的培养液上清;泳道5:含有pYGT17-IL2的酵母细胞的培养液上清;泳道6:含有pYGT25-IL2的酵母细胞的培养液上清;泳道7:含有pYGT19-IL2的酵母细胞的培养液上清;泳道8:含有pYGT18-IL2的酵母细胞的培养液上清;泳道9:含有pYGT27-IL2的酵母细胞的培养液上清)。
图19显示了用于人IL32α分泌的TFP选择方法中获得的38个酵母转化细胞的培养液上清的SDS-PAGE结果(泳道M:蛋白质大小标记;泳道N:作为阴性对照的未转化细胞;泳道1-38:酵母转化细胞)。
图20显示了分泌人IL32α的酵母细胞培养液上清的SDS-PAGE和蛋白质印迹的结果(泳道M:蛋白质大小标记;泳道1:含有pYGT3-IL32α的酵母细胞的培养液上清;泳道2:含有pYGT21-IL32α的酵母细胞的培养液上清;泳道3:含有pYGT13-IL32α的酵母细胞的培养液上清;泳道4:含有pYGT25-IL32α的酵母细胞的培养液上清;泳道5:含有pYGT22-IL32α的酵母细胞的培养液上清和泳道6:含有pYGT11-IL32α的酵母细胞的培养液上清)。
图21显示了(A)含有pYGT3-hIL32α的重组酵母菌株的分批补料发酵图和(B)分析根据发酵时间分泌至培养基中的蛋白质的SDS-PAGE结果。
图22显示了分泌人生长激素的酵母细胞的培养液上清的SDS-PAGE结果(泳道M:蛋白质大小标记;泳道N:作为阴性对照的未转化酵母细胞的培养液上清;泳道1:含有pYGT1-hGH的酵母细胞的培养液上清;泳道2:pYGT2-hGH;泳道3:pYGT3-hGH;泳道4:pYGT4-hGH;泳道5:pYGT5-hGH;泳道6:pYGT6-hGH;泳道7:pYGT7-hGH;泳道8:pYGT8-hGH;泳道9:pYGT9-hGH;泳道10:pYGT21-hGH;泳道11:pYGT13-hGH;泳道12:pYGT25-hGH;泳道13:pYGT17-hGH;泳道14:pYGT22-hGH;泳道15:pYGT32-hGH;泳道16:pYGT19-hGH;泳道17:pYGT27-hGH;泳道18:pYGT11-hGH;泳道19:pYGT40-hGH;泳道20:pYGT43-hGH;泳道21:pYGT44-hGH)。
图23显示了(A)含有pYGT18-hGH的重组酵母菌株的分批补料发酵的图和(B)分析根据发酵时间分泌至培养基中的蛋白质的SDS-PAGE结果。
图24显示了使用单向缺失方法从选择的ORFs中构建TFP文库的方法。
图25显示了随机选择的酵母转化细胞的培养液上清的SDS-PAGE结果,所述酵母转化细胞用单向缺失的TFP文库转化,其中该单向缺失的TFP文库用通过BLAST搜索选择的ORFs构建。
图26显示了随机选择的酵母转化细胞的培养液上清的SDS-PAGE结果,所述酵母转化细胞用单向缺失的TFP文库转化的,其中该单向缺失的TFP文库由被选择的35个ORFs构建。
图27显示了分泌人胰岛素样生长因子的酵母细胞的培养液上清的SDS-PAGE和蛋白质印迹(抗hIGF)结果(泳道M:蛋白质大小标记;泳道1:含有pYGa-MFa-hIGF的酵母细胞培养液上清;泳道2:pYGa-T1α-IGF;泳道3:pYGa-T2α-IGF;泳道4:pYGa-T3α-IGF;泳道5:pYGa-T4α-IGF)。
图28显示了用TFP载体转化以分泌人胱天蛋白酶-1亚基P10的酵母细胞的培养液上清的SDS-PAGE结果(泳道M:蛋白质大小标记;泳道1:含有pYGT1-hP10的酵母细胞的培养液上清;泳道2:pYGT2-hP10;泳道3:pYGT3-hP10;泳道4:pYGT4-hP10;泳道5:pYGT5-hP10;泳道6:pYGT6-hP10;泳道7:pYGT7-hP10;泳道8:pYGT8-hP10;泳道9:pYGT9-hP10;泳道10:pYGT21-hP10;泳道11:pYGT13-hP10;泳道12:pYGT25-hP10;泳道13:pYGT17-hP10;泳道16:pYGT22-hP10;泳道18:pYGT18-hP10;泳道19:pYGT33-hP10;泳道20:pYGT19-hP10;泳道21:pYGT27-hP10;泳道22:pYGT11-hP10;泳道24:pYGT39-hP10;泳道25:pYGT40-hP10;泳道28:pYGT43-hP10;泳道29:pYGT44-hP10;泳道32:阴性对照)。
图29显示了分泌人白细胞介素32γ的酵母细胞的培养液上清的SDS-PAGE和蛋白质印迹(抗IL32)结果(泳道M:蛋白质大小标记;泳道C:作为阴性对照的未转化酵母细胞的培养液上清;泳道1:pYGT1-IL32γ;泳道2:pYGT2-IL32γ;泳道3:pYGT3-IL32γ;泳道4:pYGT4-IL32γ;泳道5:pYGT5-IL32γ;泳道6:pYGT6-IL32γ;泳道7:pYGT7-IL32γ;泳道8:pYGT8-IL32γ;泳道9:pYGT9-IL32γ;泳道10:pYGT21-IL32γ;泳道11:pYGT13-IL32γ;泳道12:pYGT25-IL32γ;泳道13:pYGT17-IL32γ;泳道16:pYGT22-IL32γ;泳道18:pYGT18-IL32γ;泳道19:pYGT33-IL32γ;泳道20:pYGT19-IL32γ;泳道21:pYGT27-IL32γ;泳道22:pYGT11-IL32γ;泳道24:pYGT39-IL32γ;泳道25:pYGT40-IL32γ;泳道28:pYGT43-IL32γ;泳道29:pYGT44-IL32γ;泳道33:pYGT48-IL32γ;泳道35:pYGT50-IL32γ;泳道36:pYGT51-IL32γ;泳道37:pYGT52-IL32γ;泳道39:pYGT54-IL32γ)。
图30显示了分泌人白细胞介素-2的酵母细胞的培养液上清的SDS-PAGE结果(泳道M:蛋白质大小标记;泳道1:含有YGaSW-pSUN-IL2的酵母细胞的培养液上清;泳道2:YGaSW-pSED-IL2;泳道3:YGaSW-pUNK-IL2;泳道4:YGaSW-pMUC-IL2)。
发明详述
本发明满足了鉴定TFP的快速和有效的筛选技术的需求,特别适用于可最大分泌靶蛋白的靶蛋白。本发明对优化任何蛋白质的重组表达有效,尤其对由于在已知表达系统中低水平表达而不能大规模和/或低成本制备的蛋白质的制备有效。
在一个实施方案中,本发明涉及鉴定靶蛋白特异的TFP的方法,所述方法包含:
(i)使用多个线性载体和编码靶蛋白的核苷酸序列共转化多个报告蛋白缺乏的宿主细胞来制备多个转化的宿主细胞,
其中每个所述的线性载体包含一个来自于核酸片段文库中的核酸片段和编码N末端氨基酸缺失的报告蛋白的核苷酸序列,和
其中所述编码靶蛋白的核苷酸序列在3’末端包含一个编码N末端氨基酸的核苷酸序列,其中在所述线性载体内所述报告蛋白上缺乏该N末端氨基酸;和在5’末端包含一个连接DNA;
(ii)在允许所述线性载体和所述编码靶蛋白的核苷酸序列体内重组的有效条件下培养多个所述转化的宿主细胞;
(iii)从(ii)中的多个转化的宿主细胞中鉴定显示报告蛋白活性的细胞;和
(iv)从(iii)中鉴定的细胞中鉴定TFP;
其中所述TFP包含诱导所述靶蛋白分泌的核酸片段。
本发明的另一个实施方案涉及鉴定靶蛋白特异的TFP文库的方法,所述方法包含:
(i)用多个线性载体和编码靶蛋白的核苷酸序列共转化多个报告蛋白缺乏的宿主细胞来制备多个转化的宿主细胞,
其中每个所述线性载体包含一个来自于核酸片段文库的核酸片段和一个编码N末端氨基酸缺失的报告蛋白的核苷酸序列,和
其中所述编码靶蛋白的核苷酸序列在3’末端包含一个编码N末端氨基酸缺失的核苷酸序列,其中在所述线性载体内所述报告蛋白上缺乏该N末端氨基酸;和在5’末端包含一个连接DNA;
(ii)在允许所述线性载体和所述编码靶蛋白的核苷酸序列体内重组的有效条件下培养多个所述转化的宿主细胞;
(iii)从(ii)中的多个转化的宿主细胞中鉴定显示报告蛋白活性的细胞;和
(iv)从(iii)中鉴定的细胞中鉴定TFP文库;
其中所述TFP文库包含单独诱导所述靶蛋白分泌的核酸片段。
核酸片段文库可从任何类型的DNA,包括基因组DNA、cDNA、合成DNA和重组DNA中获得。除DNA外的核酸也可使用,包括RNA和非天然产生的核酸。
可从任何真核或原核生物,包括细菌、真菌(例如酵母)、植物和动物(例如哺乳动物)中的DNA中鉴定TFPs。适合的细菌包括,但是不限于,埃希菌和杆菌属。适合的酵母包括,但是不限于,念珠菌属、德巴利酵母属、汉森酵母属、克鲁维酵母属、毕赤酵母属、裂殖酵母属、耶罗威亚酵母属、酵母菌、许旺酵母属和Arxula属。具体种属的实例包括产朊假丝酵母、博伊丁假丝酵母、白色念珠菌、产乳糖酶酵母、巴斯德毕赤酵母、木糖发酵酵母、粟酒裂殖酵母、酿酒酵母、多形汉森酵母、解脂耶氏酵母、西方许旺酵母和Arxula adeninivorans。其它可作为DNA来源的真菌包括,但是不限于,曲霉属、青霉属、根霉属和木霉属。可作为DNA来源的植物包括,但是不限于,拟南芥、玉米、烟草和马铃薯。适合的动物包括,但是不限于,人、小鼠、大鼠、兔、狗、猫和猴。
核酸片段可来源于生物体的完整基因组,例如完整基因组或cDNA文库。这些片段还可来源于完整基因组的任何亚群,例如扣除文库或限量文库(sized library)。
在一个实施方案中,核酸片段来源于预先选定的候选TFPs文库,例如包含已在先前筛选中鉴定了的TFPs文库。在一个具体的实施方案中,预先选定的候选TFPs文库是已鉴定为对一个或多个靶蛋白有效TFPs的核心TFPs文库。
在另一个实施方案中,预先选定的候选TFPs文库通过用多种包含核酸片段的文库和编码报告蛋白的核酸序列的载体转化多个报告蛋白缺乏的宿主细胞、收集生长的细胞、从细胞中分离出载体和从载体中分离出核酸片段,从而获得包含单独诱导报告蛋白分泌的核酸片段的TFP文库。
在另一个实施方案中,预先选定的候选TFPs文库来源于在基因组数据库中鉴定了的序列,通过搜索(i)含有与那些预先鉴定了的一个或多个TFPs同源的前分泌(pre-secretion)信号的基因;(ii)包含分泌信号序列的基因;或(iii)编码通过内质网的蛋白质的基因(例如细胞壁蛋白质、分泌蛋白质、质膜蛋白质、空泡蛋白质、芽(bud)蛋白质)。
在另一个实施方案中,通过改变预先鉴定了的TFPs,例如通过单向缺失、突变、添加功能序列(例如糖基化位点)或TFPs间交换前(pre)和原(pro)信号序列获得预先选定的候选TFPs文库。
在一个实施方案中,核酸片段的大小少于1000个碱基对,例如少于700、500或300个碱基对。在另一个实施方案中,核酸片段文库通过酶裂解DNA、cDNA合成或重组DNA技术(例如单向缺失、诱变作用)构建。
本发明的线性载体可以是在被选择的宿主细胞中有功能的任何载体。如本文所用,术语“载体”涉及能够将另一个核酸运送至其连接位置的核酸分子。一种载体是“质粒”,它是指能连接其它DNA片段的圆形双链DNA环。另一种载体是病毒载体,其中其它DNA片段可连接到该病毒基因组中。某些载体能够在其引入的宿主细胞中自主复制(例如具有细菌的复制起始区的细菌载体和附加型哺乳动物载体)。其它载体(例如非附加型哺乳动物载体)一旦引入至宿主细胞后就整合至宿主细胞的基因组中从而随着宿主基因组复制。本发明的载体能够指导编码与它们可操作性连接的靶蛋白的基因的表达。这些载体本文中称为“表达载体”。一般而言,在重组DNA技术中有用的表达载体通常是质粒的形式。在本说明书中,“质粒”和“载体”可互换使用因为质粒是载体的最常用形式。但是,本发明也包括具有相同功能的其它形式的表达载体,例如病毒载体(例如复制缺陷的逆转录病毒、腺病毒和腺伴随病毒)。
原核生物中蛋白质的表达可用含有组成或诱导型启动子的载体进行,它们能指导靶蛋白-报告蛋白融合的表达。适合的大肠杆菌表达载体的实例包括pTrc(Amarann等人,Gene 69:301-315(1988))和pET(Studier等人,GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185,Academic Press,San Diego,Calif.(1990)60-89)。
为了在酵母细胞中表达,适合的酵母表达载体包括,但是不限于,pYepSecl(Bladari等人,EMBO J.6:229-234(1987))、pMFa(Kurjan等人,Cell 30:933-943(1982))、pJRY88(Schultz等人,Gene 54:113-123(1987))、pYES2(Invitrogen Corporation,San Diego,Calif.)和picZ(Invitrogen Corp.San Diego,Cal.)。
为了在昆虫细胞中表达,可使用杆状病毒表达载体。可用于在培养的昆虫细胞(例如SG9细胞)中表达蛋白质的杆状病毒载体包括pAc系列(Smith等人,Mol.Cell.Biol.3:2156-2165(1983))和pVL系列(Lucklow等人,Virology 170:31-39(1989))。
在另一个实施方案中,宿主细胞是哺乳动物细胞而载体是哺乳动物表达载体。哺乳动物表达载体的实例包括pCDM8(Seed,Nature 329:840(1987))和pMT2PC(Kaufman等人,EMBO J.6:187-195(1987))。当用于哺乳动物细胞时,表达载体的调控功能常常由病毒调控元件提供。例如,通常使用的启动子来源于多瘤腺病毒2、巨细胞病毒和猿猴病毒40。其它适合的用于原核和真核细胞两者的表达系统参见例如Sambrook等人,MOLECULAR CLONING:A LABORATORY MANUAL.第二版,Cold Spring HarborLaboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989,第16和17章。
优选的载体包括但不限于质粒、噬菌体、粘粒、游离基因、病毒颗粒或病毒和整合的DNA片段(例如通过同源重组整合至宿主基因组的片段)。优选的病毒颗粒包括但不限于腺病毒、杆状病毒、细小病毒、疱疹病毒、痘病毒、腺相关病毒、塞姆利基森林病毒、痘苗病毒和逆转录病毒。优选的表达载体包括但不限于pcDNA3(Invitrogen)和pSVL(Pharmacia Biotech)。其它表达载体包括但不限于pSPORTTM载体、pGEMTM载体(Promega)、pPROEX载体TM(LTI,Bethesda,MD)、BluescriptTM载体(Stratagene)、pQETM载体(Qiagen)、pSE420TM(Invitrogen)和pYES2TM(Invitrogen)。
在一个实施方案中,表达载体是复制型DNA构建体,其中编码靶蛋白的DNA序列可操作性的连接至能够影响靶蛋白在适合的宿主中表达的适合的调控序列中。当它们的功能彼此相关时DNA区域可操作性的连接。例如,启动子可操作性的连接于编码序列如果该启动子能调控该编码序列的转录。扩增载体不需要表达调控域而更需要在宿主中进行复制的能力,这通常由复制起始区决定和帮助转化体识别的选择基因。表达载体对调控序列的需要取决于选择的宿主和选择的转化方法。一般来说,调控序列包括但不限于转录启动子、增强子、控制转录的可选操纵子序列、多腺苷酸化信号、编码适合的结合核糖体的mRNA的序列和调控转录和翻译终止的序列。这些调控序列描述于例如Goeddel,GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185,Academic Press,San Diego,Calif.(1990)。调控序列包括那些指导多种宿主细胞中的核苷酸序列组成型表达和那些仅在特定宿主细胞中指导核苷酸序列表达(例如组织特异性调控能够序列)的调控序列。本领域技术人员应当理解表达载体的设计取决于所选择的要转化的宿主细胞、预期的蛋白质表达的水平等等这些因素。
本发明的表达载体可引入宿主细胞从而制备蛋白质或肽,包括本文描述的核酸所编码的融合蛋白或肽。优选的载体优选含有宿主生物体识别的启动子。本发明的启动子序列可以是原核、真核或病毒的。适合的原核序列的实例包括λ噬菌体的PR和PL启动子(The bacteriophage Lambda,Hershey,A D.,Ed.,Cold Spring Harbor Press,Cold SpringHarbor,NY(1973),将其全文引入本文作为参考;Lambda II,Hendrix,R.W.,Ed.,ColdSpring Harbor Press,Cold Spring Harbor,NY(1980),将其全文引入本文作为参考);大肠杆菌的trp、recA、热休克和lacZ启动子以及SV40早期启动子(Benoist等人,Nature,290:304-310(1981),将其全文引入本文作为参考)。对于酵母,适合的启动子的实例包括但不限于GAPDH、PGK、ADH、PHO5、GAL1和GAL10。其它启动子包括但不限于小鼠乳腺瘤病毒、人免疫缺陷症病毒长末端复制、maloney病毒、巨细胞病毒立即早期启动子、EB病毒(Epstein Bar virus)、劳斯肉瘤病毒、人肌动蛋白、人肌球蛋白、人血红蛋白、人肌酸和人金属硫蛋白。
其它调控序列还可包括在优选载体中。适合的调控序列的实例是噬菌体MS-2的复制酶基因和λ噬菌体的cII基因的夏因-达尔加诺序列。
此外,适合的表达载体可包括允许筛选转化的宿主细胞的适当标记。选择的宿主的转化使用各种本领域技术人员公知和先前Sambrook等人描述的技术中的任意一种来进行。
复制起始区还可通过构建载体以包括外源的起始区来提供或通过宿主细胞染色体复制机制提供。如果将载体整合入宿主细胞染色体,后者就足够了。另外,相比使用含有病毒复制起始区的载体,本领域技术人员可使用用选择性标记和靶蛋白DNA共转化的方法来转化哺乳动物细胞。适合的标记的一个实例是二氢叶酸还原酶(DHFR)或胸苷激酶(参见美国专利4,399,216)。
编码靶蛋白的核苷酸序列可用载体DNA利用常规技术重组,包括平末端或粘末端(staggered)连接、限制性酶消化来提供适当的末端、适当补平粘性末端、碱性磷酸酶处理以避免非预期的连接以及用适当连接酶连接。这些操作的技术先前由Sambrook等人公开且在本领域内公知。构建哺乳动物表达载体的方法公开于例如Okayama等人,Mol.Cell.Biol.3:280(1983)、Cosman等人,Mol.Immunol.23:935(1986)、Cosman等人,Nature 312:768(1984)、EP-A-0367566和WO 91/18982,每篇均全文引入本文作为参考。
本发明所用的宿主细胞可以是本领域技术人员公知的任何宿主细胞。适合的宿主细胞包括细菌、真菌、(例如酵母)、植物或动物(例如哺乳动物或昆虫)细胞。适合的酵母细胞包括念珠菌属、德巴利酵母属、汉森酵母属、克鲁维酵母属、毕赤酵母属、裂殖酵母属、耶罗威亚酵母属、酵母菌、许旺酵母属和Arxula属。具体实例包括产朊假丝酵母、博伊丁假丝酵母、白色念珠菌、产乳糖酶酵母、巴斯德毕赤酵母、木糖发酵酵母、粟酒裂殖酵母、酿酒酵母、多形汉森酵母、解脂耶氏酵母、西方许旺酵母和Arxulaadeninivorans。其它适合的的真菌包括曲霉属、青霉属、根霉属和木霉属。可用作宿主细胞的细菌包括埃希菌属、假单胞杆菌属和杆菌属。适合的植物宿主细胞包括拟南芥、玉米、烟草和马铃薯。动物细胞包括人、小鼠、大鼠、兔、狗、猫、猴和昆虫。实例包括CHO、COS 1、COS 7、BSC 1、BSC 40、BMT 10和Sf9细胞。
在一个具体的实施方案中,宿主细胞是酵母细胞,核酸片段分离自酵母的基因组或cDNA。
本发明的多聚核苷酸可引入宿主细胞作为环状质粒的部分或作为包含分离蛋白质编码区域的线性DNA或病毒载体。本领域众所周知且常规进行的引入DNA至宿主细胞的方法包括转化、转染、电穿孔法、核注射或与载体例如脂质体、胶束、血影细胞和原生质体融合。
任何可快速有效检测的报告蛋白可用于本发明。在一个实施方案中,为了使筛选过程自动化,报告蛋白具有确定选择的活性。在另一个实施方案中,报告蛋白是分泌至细胞外间隙的蛋白质,例如转化酶、蔗糖酶、纤维素酶、木聚糖酶、麦芽糖酶、淀粉酶、葡萄糖淀粉酶、半乳糖苷酶(例如α-半乳糖苷酶、β-半乳糖苷酶、蜜二糖酶)、磷酸酶(例如PHO5)、β-内酰胺酶、脂肪酶或蛋白酶。在一个具体的实施方案中,分泌蛋白允许细胞在特定底物上生长。作为哺乳动物细胞中的报告基因系统的实例,CD2/新霉素-磷酸转移酶(Ceo)基因可用作含有抗生素G418的培养基中的分泌报告基因以捕获在小鼠胚胎干细胞中分泌通路基因(De-Zolt等人,Nucleic Acid Res.34:e25(2006))。
在一个实施方案中,宿主细胞是酵母,报告蛋白是转化酶,转化的酵母细胞根据它们在蔗糖或蜜三糖上生长的能力来选择。在另一个实施方案中,宿主细胞是酵母,报告蛋白是蜜二糖酶,转化的酵母细胞根据它们在蜜二糖上生长的能力来选择。在另一个实施方案中,宿主细胞是酵母,报告蛋白是淀粉酶(例如内淀粉酶、外淀粉酶、β-淀粉酶或葡萄糖淀粉酶),酵母细胞是非淀粉分解的,转化的细胞根据它们降解淀粉的能力来筛选。在另一个实施方案中,鉴定显示报告蛋白活性的细胞的步骤通过使用具有生长抑制因子抗性的报告蛋白来进行,例如抗生素。在另一个实施方案中,报告蛋白是能够视觉上检测的蛋白质,例如绿色荧光蛋白或荧光素酶。在一个实施方案中,鉴定显示报告蛋白活性的细胞的步骤通过使用两个或多个报告蛋白例如脂肪酶和转化酶来进行。
本发明的宿主细胞不显示报告蛋白活性。在一个实施方案中,宿主细胞不天然表达报告蛋白。在其它实施方案中,编码报告蛋白的基因(们)已被全部或部分删除或已突变以使得报告蛋白不表达或以无活性形式表达。使一个细胞缺乏一种特殊的蛋白质的方法在本领域内公知,并且任何此种方法可用来制备本发明的宿主细胞(前述Sambrook等人)。对于酵母来说,报告蛋白缺乏可使用公知的基因置换技术来引入(Rothstein,Meth.Enzymol.194:281(1991))。
本发明的线性载体包含核酸片段和编码N末端氨基酸缺失的报告蛋白的核苷酸序列。N末端氨基酸缺失可包含任意数目的氨基酸只要该缺失足够基本上消除报告蛋白活性即可。例如,缺失可在报告蛋白N末端包含大约5、10、15、20、25、30、35、40、45或50或更多氨基酸。
本发明的方法可使用预期高水平重组表达的任何靶蛋白。靶蛋白可以是用来进行研究目的或为商业目的所制备的,例如治疗或工业用途。靶蛋白可来自于任何植物、动物或微生物,可天然产生或以任何途径修饰,只要它能被核酸编码即可。在一个实施方案中,靶蛋白是人蛋白质。在另一个实施方案中,靶蛋白是细胞因子、血清蛋白、集落刺激因子、生长因子、激素或酶。例如,靶蛋白可选自白细胞介素、凝结因子、干扰素-α、-β或-γ、粒细胞集落刺激因子、粒细胞巨噬细胞集落刺激因子、组织生长因子、上皮生长因子、TGFα、TGFβ、表皮生长因子、血小板衍生生长因子、成纤维细胞生长因子、促卵泡激素、促甲状腺激素、血管升压素、色素性激素、甲状旁腺激素、促黄体生成激素释放激素、碳水化合物特异性酶、蛋白水解酶、脂肪酶、氧化还原酶、转移酶、水解酶、裂解酶、异构酶、连接酶、免疫球蛋白、细胞因子受体、乳铁蛋白、磷脂酶A2激活蛋白、胰岛素、肿瘤坏死因子、降钙素、降钙素基因相关肽、脑啡肽、生长调节素、促红细胞生成素、下丘脑释放因子、催乳素、绒毛膜促性腺激素、组织纤溶酶原激活物、生长激素释放肽、胸腺体液因子、抗癌肽或抗菌肽。具体实例包括但不限于人白细胞介素-2、人白细胞介素-1β、人白细胞介素-6、人白细胞介素-32α、-32β或-32γ、VII因子、VIII因子、IX因子、人血清白蛋白、人干扰素-α、-β或-γ、人粒细胞集落刺激因子、人粒细胞巨噬细胞集落刺激因子、人生长激素、人血小板衍生生长因子、人碱性成纤维细胞生长因子、人表皮生长因子、人胰岛素样生长因子、人神经生长因子、人转化生长因子β-1、人促卵泡激素、葡萄糖氧化酶、glucodase、半乳糖苷酶、葡糖脑苷脂酶、葡糖醛酸糖苷酶、门冬酰胺酶、精氨酸酶、精氨酸脱氨基酶、过氧化物歧化酶、endotoxinase、过氧化氢酶、糜蛋白酶、尿酸酶、腺苷二磷酸酶、酪氨酸酶、胆红素氧化酶、牛1-磷酸半乳糖尿甘酸转移酶、水母绿色荧光蛋白、南极假丝酵母脂肪酶B、假丝酵母脂肪酶、真菌氯过氧化物酶、β-半乳糖苷酶、解离酶、α-半乳糖苷酶、β-葡萄糖苷酶、海藻糖合酶、环糊精糖基转移酶、木聚糖酶、植酸酶、人乳铁蛋白、人促红细胞生成素、人对氧磷酶、人生长分化因子15、人半乳凝素-3结合蛋白、人丝氨酸蛋白酶抑制剂、Kunitz2型、人Janus激酶2、人fms-like酪氨酸激酶3配体、人YM1&2、人CEMI、人二酰基甘油酰基转移酶、人瘦蛋白、人mL259、人蛋白水解酶3、人溶菌酶、人DEAD box蛋白41、人依托泊苷诱导蛋白24、鼠细胞凋亡蛋白酶1、牛血管生成因子和蚯蚓蚓激酶。
在一个实施方案中,靶蛋白是用常规重组生产方法难以生产的蛋白质,即根本不生产或仅低水平生产的蛋白质。在另一个实施方案中,靶蛋白是使用已知表达系统容易产生但希望获得较高水平表达的蛋白质。
编码靶蛋白的核酸可使用本领域公知的常规技术从任何来源获得,包括从基因组或cDNA文库中分离出来、PCR扩增或化学合成。
本发明的方法使用的编码靶蛋白的核苷酸序列在5’末端包含连接DNA,该连接DNA用于用本发明的线性载体进行体内重组,并在3’末端包含编码报告蛋白N末端部分的核苷酸序列,包括线性载体缺失的N末端氨基酸和足够的其它氨基酸以允许编码靶蛋白的核苷酸序列和线性载体在共转化至宿主细胞时在体内重组。在一个实施方案中,编码报告蛋白N末端部分的序列包含至少20个与线性载体内编码报告蛋白的序列重叠的碱基对,例如至少30或40个碱基对。将5’连接子和3’报告蛋白序列加入至编码靶蛋白的核苷酸序列可使用常规重组DNA技术来进行,例如PCR和/或限制性酶切割和连接。
本发明的连接DNA必须有足够长度和足够与线性载体的部分核苷酸序列相同的序列以允许编码靶蛋白的核苷酸序列和线性载体在共转化至宿主细胞时在体内重组。在一个实施方案中,连接DNA的长度超过20个碱基对,例如长度超过30或40个碱基对。在另一个实施方案中,连接DNA与线性载体上的相应序列有至少80%相同,例如至少85%、90%、95%或99%相同。
在一个实施方案中,连接DNA编码蛋白酶识别序列从而允许在TFP和靶蛋白的接点处断裂。例如,连接DNA可编码酵母样kex2p-或Kex2-蛋白酶识别序列(例如包含Lys-Arg、Arg-Arg或Leu-Asp-Lys-Arg(SEQ ID NO:214)的氨基酸序列)、哺乳动物弗林(furin)蛋白酶识别序列(例如包含Arg-X-X-Arg的氨基酸序列)、Xa因子识别序列(例如包含Ile-Glu-Gly-Arg(SEQ ID NO:215)的氨基酸序列)、肠激酶识别序列(例如包含Asp-Asp-Lys的氨基酸序列)、枯草杆菌蛋白酶识别序列(例如包含Ala-Ala-His-Tyr(SEQID NO:216)的氨基酸序列)、烟草蚀纹病毒蛋白酶识别序列(例如包含Glu-Asn-Leu-Tyr-Phe-Gln-Gly(SEQ ID NO:217)的氨基酸序列)、遍在蛋白质水解酶识别序列(例如包含Arg-Gly-Gly的氨基酸序列)或凝血酶识别序列(例如包含Arg-Gly-Pro-Arg(SEQ ID NO:218)的氨基酸序列)。
在另一个实施方案中,连接DNA编码亲合标签、例如GST、MBP、NusA、硫氧还蛋白、遍在蛋白质、FLAG、BAP、6HIS、STREP、CBP、CBD或S-标签。
在另一个实施方案中,连接DNA编码限制性酶识别位点,例如SfiI位点。在另一个实施方案中,连接DNA编码限制性酶识别位点和蛋白酶识别序列(例如类kex2p-蛋白酶-或kex-2p-识别序列)。
本发明涉及通过本发明的方法鉴定的TFP或其衍生物或片段。在一个实施方案中,TFP选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ ID NO:33)、TFP-18(SEQ ID NO:35)、TFP-19(SEQ ID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQ ID NO:61)、TFP-22(SEQ ID NO:63)、TFP-29(SEQ ID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQID NO:69)、TFP-39(SEQ ID NO:129)、TFP-43(SEQ ID NO:131)、TFP-44(SEQ ID NO:133)、TFP-48(SEQ ID NO:135)、TFP-52(SEQ ID NO:137)、TFP-54(SEQ ID NO:139)、TFP-40(SEQ ID NO:175)、TFP-50(SEQ ID NO:177)、TFP-51(SEQ ID NO:179)、TFP-57(SEQ ID NO:181)、TFP-58(SEQ ID NO:183)、TFP-59(SEQ ID NO:185)、TFP-5(SEQID NO:200)、TFP-6(SEQ ID NO:202)、TFP-7(SEQ ID NO:204)、TFP-8(SEQ ID NO:206)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ ID NO:86)、PpTFP-3(SEQ ID NO:88)和PpTFP-4(SEQ ID NO:90)组成的组或其衍生物或片段。
本发明还涉及TFP文库,该TFP文库包含两种或多种通过本发明的方法鉴定的TFPs或其片段或衍生物。在一个实施方案中,TFP文库包含鉴定为对特殊靶蛋白有效的TFPs。在另一个实施方案中,TFP文库包含鉴定为对超过一个靶蛋白有效的TFPs。在一个具体的实施方案中,TFP文库包含两个或更多(例如4、6、8、10或12或更多)选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ ID NO:33)、TFP-18(SEQ IDNO:35)、TFP-19(SEQ ID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQ ID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQ ID NO:61)、TFP-22(SEQID NO:63)、TFP-29(SEQ ID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQ ID NO:69)、TFP-39(SEQ ID NO:129)、TFP-43(SEQ ID NO:131)、TFP-44(SEQ ID NO:133)、TFP-48(SEQ ID NO:135)、TFP-52(SEQ ID NO:137)、TFP-54(SEQ ID NO:139)、TFP-40(SEQ IDNO:175)、TFP-50(SEQ ID NO:177)、TFP-51(SEQ ID NO:179)、TFP-57(SEQ ID NO:181)、TFP-58(SEQ ID NO:183)、TFP-59(SEQ ID NO:185)、TFP-5(SEQ ID NO:200)、TFP-6(SEQID NO:202)、TFP-7(SEQ ID NO:204)、TFP-8(SEQ ID NO:206)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ ID NO:86)、PpTFP-3(SEQ ID NO:88)和PpTFP-4(SEQ ID NO:90)组成的组或其衍生物或片段。
在另一个实施方案中,TFP文库包含六个或更多(例如8、10、12或15或更多)选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ ID NO:33)、TFP-18(SEQID NO:35)、TFP-19(SEQ ID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQ ID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQ ID NO:61)、TFP-22(SEQID NO:63)、TFP-29(SEQ ID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQ ID NO:69)、TFP-39(SEQ ID NO:129)、TFP-43(SEQ ID NO:131)、TFP-44(SEQ ID NO:133)、TFP-48(SEQ ID NO:135)、TFP-52(SEQ ID NO:137)、TFP-54(SEQ ID NO:139)、TFP-40(SEQ IDNO:175)、TFP-50(SEQ ID NO:177)、TFP-51(SEQ ID NO:179)、TFP-57(SEQ ID NO:181)、TFP-58(SEQ ID NO:183)、TFP-59(SEQ ID NO:185)、TFP-5(SEQ ID NO:200)、TFP-6(SEQID NO:202)、TFP-7(SEQ ID NO:204)、TFP-8(SEQ ID NO:206)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ ID NO:86)、PpTFP-3(SEQ ID NO:88)、PpTFP-4(SEQ ID NO:90)、TFP-1(SEQ ID NO:219)、TFP-2(SEQ ID NO:221)、TFP-3(SEQ ID NO:223)、TFP-4(SEQ IDNO:225)和TFP 32(SEQ ID NO:208)组成的组的TFPs或其衍生物或片段。
本发明还涉及编码用本发明的方法鉴定的编码TFP或其衍生物或片段的核酸。在一个实施方案中,核酸编码的TFP选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ ID NO:33)、TFP-18(SEQ ID NO:35)、TFP-19(SEQ ID NO:37)、TFP-20(SEQID NO:39)、TFP-21(SEQ ID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQ ID NO:61)、TFP-22(SEQ ID NO:63)、TFP-29(SEQ ID NO:65)、TFP-34(SEQID NO:67)、TFP-38(SEQ ID NO:69)、TFP-39(SEQ ID NO:129)、TFP-43(SEQ ID NO:131)、TFP-44(SEQ ID NO:133)、TFP-48(SEQ ID NO:135)、TFP-52(SEQ ID NO:137)、TFP-54(SEQ ID NO:139)、TFP-40(SEQ ID NO:175)、TFP-50(SEQ ID NO:177)、TFP-51(SEQ ID NO:179)、TFP-57(SEQ ID NO:181)、TFP-58(SEQ ID NO:183)、TFP-59(SEQ ID NO:185)、TFP-5(SEQ ID NO:200)、TFP-6(SEQ ID NO:202)、TFP-7(SEQID NO:204)、TFP-8(SEQ ID NO:206)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ IDNO:86)、PpTFP-3(SEQ ID NO:88)和PpTFP-4(SEQ ID NO:90)组成的组或其衍生物或片段。在一个实施方案中,核酸包含选自由SEQ IDNOS:30、32、34、36、38、40、42、44、46、62、64、66、68、70、85、87、89、91、130、132、134、136、138、140、176、178、180、182、184、186、201、203、205或207组成的组的核苷酸序列或其衍生物或片段。
本发明还涉及编码两个或多个用本发明的方法鉴定的TFPs或其衍生物或片段的核酸文库。在一个实施方案中,该核酸文库编码鉴定为对特殊靶蛋白有效的TFPs。在另一个实施方案中,该核酸文库编码鉴定为对超过一个靶蛋白有效的TFPs。在一个具体的实施方案中,该核酸文库编码两个或更多TFPs(例如4、6、8、10或12或更多),选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ ID NO:33)、TFP-18(SEQ ID NO:35)、TFP-19(SEQ ID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQ ID NO:61)、TFP-22(SEQ ID NO:63)、TFP-29(SEQ ID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQID NO:69)、TFP-39(SEQ ID NO:129)、TFP-43(SEQ ID NO:131)、TFP-44(SEQ ID NO:133)、TFP-48(SEQ ID NO:135)、TFP-52(SEQ ID NO:137)、TFP-54(SEQ ID NO:139)、TFP-40(SEQ ID NO:175)、TFP-50(SEQ ID NO:177)、TFP-5I(SEQ ID NO:179)、TFP-57(SEQ ID NO:181)、TFP-58(SEQ ID NO:183)、TFP-59(SEQ ID NO:185)、TFP-5(SEQID NO:200)、TFP-6(SEQ ID NO:202)、TFP-7(SEQ ID NO:204)、TFP-8(SEQ ID NO:206)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ ID NO:86)、PpTFP-3(SEQ ID NO:88)和PpTFP-4(SEQ ID NO:90)组成的组或其衍生物或片段。在一个实施方案中,核酸文库包含两个或更多(例如4、6、8、10或12或更多)SEQ ID NOS:30、32、34、36、38、40、42、44、46、62、64、66、68、70、85、87、89、91、130、132、134、136、138、140、176、178、180、182、184、186、201、203、205或207的核苷酸序列或其衍生物或片段。
在另一个实施方案中,核酸文库编码六个或更多(例如8、10、12或15或更多)TFPs,该TFPs选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ ID NO:33)、TFP-18(SEQ ID NO:35)、TFP-19(SEQ ID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQ ID NO:61)、TFP-22(SEQ ID NO:63)、TFP-29(SEQ ID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQID NO:69)、TFP-39(SEQ ID NO:129)、TFP-43(SEQ ID NO:131)、TFP-44(SEQ IDNO:133)、TFP-48(SEQ ID NO:135)、TFP-52(SEQ ID NO:137)、TFP-54(SEQ ID NO:139)、TFP-40(SEQ ID NO:175)、TFP-50(SEQ ID NO:177)、TFP-51(SEQ ID NO:179)、TFP-57(SEQ ID NO:181)、TFP-58(SEQ ID NO:183)、TFP-59(SEQ ID NO:185)、TFP-5(SEQID NO:200)、TFP-6(SEQ ID NO:202)、TFP-7(SEQ ID NO:204)、TFP-8(SEQ ID NO:206)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ ID NO:86)、PpTFP-3(SEQ ID NO:88)、PpTFP-4(SEQ ID NO:90)、TFP-1(SEQ ID NO:219)、TFP-2(SEQ ID NO:221)、TFP-3(SEQID NO:223)、TFP-4(SEQ ID NO:225)和TFP 32(SEQ ID NO:208)组成的组或其衍生物或片段。在一个实施方案中,核酸文库包含六个或更多(例如8、10、12或15或更多)SEQ IDNOS:30、32、34、36、38、40、42、44、46、62、64、66、68、70、85、87、89、91、130、132、134、136、138、140、176、178、180、182、184、186、201、203、205、207、209、220、222、224或226的核苷酸序列或其衍生物或片段。
术语“其片段”,正如TFP所使用的,是指包括TFP氨基酸序列的任何部分的多肽,其中该片段基本上保留诱导与其融合的靶蛋白的分泌的能力。
术语“其衍生物”,正如TFP所使用的,是指由与TFP的氨基酸序列至少70%相同的氨基酸序列组成的多肽,其中该多肽基本上保留诱导与其融合的靶蛋白分泌的能力。在一些实施方案中,该衍生物包含与TFP的氨基酸序列至少75%、80%、85%、90%、95%、96%、97%、98%或99%相同的氨基酸序列。该衍生物还可包含对TFP氨基酸序列的增加、删除、取代或组合。增加或取代还包括使用非天然产生氨基酸。
取代优选保守氨基酸取代。“保守氨基酸取代”是指氨基酸残基用具有相似侧链的氨基酸残基代替。具有相似侧链的氨基酸残基家族本领域内已有定义。这些家族包括具有碱性侧链(例如赖氨酸、精氨酸、组氨酸),酸性侧链(例如门冬氨酸、谷氨酸)、不带电荷极性侧链(例如甘氨酸、天门冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-支链侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。
术语“其衍生物”,正如编码TFP的核酸的所使用的,是指由与编码TFP的核酸的核苷酸序列至少70%相同的核苷酸序列组成的核酸,其中该衍生物编码的多肽基本上保留了诱导与其融合的靶蛋白分泌的能力。在一些实施方案中,该衍生物包含与编码TFP的核酸的核苷酸序列至少75%、80%、85%、90%、95%、96%、97%、98%或99%相同的核苷酸序列。该衍生物可能包含编码TFP的核酸的核苷酸序列的添加、删除、取代、或其组合。
序列相同性通过比较两个在比较区域进行最佳排列的序列、确定相同氨基酸残基或核苷酸在两个序列中所处位置的数目以获得匹配位置的数目、用比较区域(例如窗口大小)的位置的总数目除匹配位置的数目并使结果乘100来获得序列相同性的百分数来计算。一方面,当长度为100个氨基酸或核苷酸的四个空位引入可使对准最大化时,相同性百分比的计算是指两个序列中的较小序列氨基酸残基或核苷酸的百分数,其中所述的两个序列以被比序列中的相同氨基酸残基或核苷酸对准(Dayhoff,in Atlas of ProteinSequence and Structure,Vol.5,p.124,National Biochemical Research Foundation,Washington,D.C.(1972),引入本文作为参考)。相同性的确定通常通过本领域已知的计算机同源性程序进行。范例程序是Gap程序(Wisconsin Sequence Analysis Package,Version 8for UNIX,Genetics Computer Group,University Research Park,Madison,WI)使用默认设置,应用Smith and Waterman算法(Adv.Appl.Math.,1981,2:482-489,全文引入本文作为参考)。
衍生物的实例包括但不限于缺失突变(例如单向缺失)、功能序列的增加(例如糖基化位点、限制性酶切位点)和在TFPs中确定的前序列或原序列的删除或增加(例如交换)。本领域技术人员可使用常规诱变技术制备TFPs衍生物或编码TFPs的核酸,例如上述引用的参考文献所描述的那些,和鉴定基本上保留诱导与其融合的靶蛋白分泌能力的衍生物。
如本文所用的术语“基本上保留诱导与其融合的靶蛋白分泌能力”是指保留原始TFP诱导与其融合的靶蛋白分泌能力至少50%的TFP片段或衍生物。在一些实施方案中,至少保留60、65、70、75、80、85、90或95%诱导与其融合的靶蛋白分泌能力。诱导靶蛋白分泌的能力可通过本领域公知和前述的常规技术来确定。
本发明的一个实施方案涉及编码TFPs的核酸片段文库,包含10个或更多由本发明的方法鉴定的核酸片段(例如50、100、500、1000或2000或更多),其中筛选中使用预先选择的候选TFPs文库。
本发明的另一个实施方案涉及编码TFPs的核酸片段文库,包含10个或更多由本发明的方法鉴定的核酸片段(例如50或100或更多),其中筛选中使用的预先选择的候选TFPs文库是通过转化多个报告蛋白缺乏的宿主细胞,收集生长的细胞、从细胞中分离出载体、并从载体中分离出核酸片段而获得的,其中所述的多个报告蛋白缺乏的宿主细胞用多种包含核酸片段的文库和编码报告蛋白的核苷酸序列进行转化,从而获得包含单独诱导报告蛋白分泌的核酸片段的TFP文库。
本发明的另一个实施方案涉及编码TFPs的核酸片段文库,包含10个或更多本发明的方法鉴定的核酸片段(例如50、100、500或1000或更多),其中用于筛选的预先选择的候选TFPs文库来自于在基因组数据库中通过搜索(i)含有与一个或多个先前鉴定的TFPs同源的前分泌信号的基因;(ii)包含分泌信号序列的基因,或(iii)编码穿过内质网的蛋白质的基因而鉴定的序列。
本发明的另一个实施方案涉及编码TFPs的核酸片段文库,包含10个或更多本发明的方法鉴定的核酸片段(例如50、100、500或更多),其中用于筛选的预先选择的候选TFPs文库通过改变先前鉴定的TFPs获得。
本发明还涉及包含编码用本发明的方法鉴定的TFP的核苷酸序列和编码靶蛋白的核苷酸序列的核酸。在一个实施方案中,TFP选自由TFP-9、TFP-13、TFP-17、TFP-18、TFP-19、TFP-20、TFP-21、TFP-25、TFP-27、TFP-11、TFP-22、TFP-29、TFP-34、TFP-38、TFP-39、TFP-43、TFP-44、TFP-48、TFP-52、TFP-54、TFP-40、TFP-50、TFP-51、TFP-57、TFP-58、TFP-59、TFP-5、TFP-6、TFP-7和TFP-8或其衍生物或片段组成的组。在另一个实施方案中,靶蛋白选自IL-2、IL-32、人生长激素和人胱天蛋白酶-1亚基P10。在一个具体的实施方案中,TFP是TFP-9、TFP-13、TFP-17、TFP-18、TFP-19、TFP-20、TFP-21、TFP-25、TFP-27、PpTFP-1、PpTFP-2、PpTFP-3、PpTFP-4或其衍生物或片段,并且靶蛋白是IL-2。在另一个实施方案中,TFP是TFP-11、TFP-22、TFP-29、TFP-34或TFP-38或其衍生物或片段,并且靶蛋白是IL-32α。在另一个实施方案中,TFP是TFP-9、TFP-13、TFP-17、TFP-18、TFP-19、TFP-20、TFP-21、TFP-25、TFP-27、TFP-11、TFP-22、TFP-29、TFP-34或TFP-38或其衍生物或片段,并且靶蛋白是生长激素。
本发明还涉及使用本发明的TFPs重组生产靶蛋白的方法。在一个实施方案中,该方法包含制备载体,所述载体包含编码靶蛋白的核苷酸序列,该核苷酸序列可操作性的连接至编码TFP或其衍生物或片段的核苷酸序列、用所述载体转化宿主细胞,并在宿主细胞生成和分泌靶蛋白的条件下培养宿主细胞。在一个实施方案中,TFP选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ ID NO:33)、TFP-18(SEQID NO:35)、TFP-19(SEQ ID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQ ID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQ ID NO:61)、TFP-22(SEQID NO:63)、TFP-29(SEQ ID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQ ID NO:69)、TFP-39(SEQ ID NO:129)、TFP-43(SEQ ID NO:131)、TFP-44(SEQ ID NO:133)、TFP-48(SEQ ID NO:135)、TFP-52(SEQ ID NO:137)、TFP-54(SEQ ID NO:139)、TFP-40(SEQ ID NO:175)、TFP-50(SEQ ID NO:177)、TFP-51(SEQ ID NO:179)、TFP-57(SEQ ID NO:181)、TFP-58(SEQ ID NO:183)、TFP-59(SEQ ID NO:185)、TFP-5(SEQID NO:200)、TFP-6(SEQ ID NO:202)、TFP-7(SEQ ID NO:204)、TFP-8(SEQ ID NO:206)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ ID NO:86)、PpTFP-3(SEQ ID NO:88)和PpTFP-4(SEQ ID NO:90)或其衍生物或片段组成的组。在另一个实施方案中,靶蛋白选自IL-2、IL-32、人生长激素和人胱天蛋白酶-1亚基P10。
靶蛋白可使用任何本领域已知表达系统重组生产。靶蛋白优选重组表达例如在细菌、酵母或哺乳动物细胞培养物中。重组表达包括制备包含编码靶蛋白的多核苷酸的载体、运送载体至宿主细胞、在靶蛋白表达的条件下培养宿主细胞和分离靶蛋白。制备重组载体和使用同样的载体转化宿主细胞、在宿主细胞中复制载体和表达生物活性外源多肽和蛋白质的方法和材料在前面讨论且描述于Sambrook等人,Molecular Cloning,第三版,Cold Spring Harbor Laboratory,2001和Ausubel等人,Current Protocols in MolecularBiology,John Wiley & Sons,New York,第三版,(2000),均引入本文作为参考。
载体DNA可通过常规转化或转染技术引入原核或真核细胞。如本文所用的术语“转化”和“转染”是指多种将外源核酸(例如DNA)引入宿主细胞的本领域认可的技术,包括磷酸钙或氯化钙共沉淀法、DEAE-葡聚糖介导的转染、脂质转染法或电穿孔法。转化或转染宿主细胞的适合方法可见于Sambrook等人(MOLECULAR CLONING:ALABORATORY MANUAL.第二版,Cold Spring Harbor Laboratory,Cold Spring HarborLaboratory Press,Cold Spring Harbor,N.Y.,1989)和其它实验手册。
为了使哺乳动物细胞稳定转染,众所周知根据使用的表达载体和转染技术,仅有很小部分的细胞可将外源DNA整合至其基因组中。为了鉴定和选择这些整合体,编码可选标记(例如抗生素抗性)的基因通常随着目的基因引入宿主细胞。多种可选标记包括那些对药物具有抗性的试剂,例如G418、潮霉素和甲氨喋呤。编码可选标记的核酸可用与编码靶蛋白相同的载体引入宿主细胞或可用单独载体引入。用引入的核酸稳定转染的细胞可用药物选择来鉴定(例如结合有可选标记基因的细胞将存活而其它细胞死亡)。
靶蛋白可从宿主细胞生长的培养基中分离出来,通过本领域已知的纯化方法例如常规色谱方法包括免疫亲和层析法、受体亲和层析法、疏水作用色谱法、凝集素亲和层析法、尺寸排阻过滤法、阳离子或阴离子交换色谱法、高压液相色谱法(HPLC)、反相HPLC等等。其它纯化方法还包括那些预期蛋白质作为融合蛋白被表达和纯化的方法,其中所述的融合蛋白具有特殊标签、标记或被特殊结合配偶体或试剂识别的螯合部分。经纯化的蛋白质可断裂以获得预期蛋白,或可作为完整的融合蛋白被保存。作为断裂过程的结果,融合组分的断裂可生成具有其它氨基酸残基的预期蛋白质形式。
如果被分离的靶蛋白在使用分离操作后没有生物活性,可使用多种方法使多肽发生“重折叠”或转化成其三级结构并生成二硫键使之恢复生物活性。本领域普通技术人员已知的方法包括在特定浓度促溶剂存在下将溶解的多肽的pH调节至通常大于7。促溶剂的选择类似于包涵体增溶作用的选择,但是通常浓度较低且不必要和增溶作用使用同种促溶剂。可能需要使用还原剂或特殊比例的还原剂和其氧化形式以得到特殊氧化还原电位使蛋白质的半胱氨酸桥形成中发生二硫链的转移。一些常用的氧化还原对包括半胱氨酸/胱胺、谷胱甘肽(GSH)/二硫代谷胱甘肽、氯化铜、二硫苏糖醇(DTT)/二噻烷DTT、2-巯基乙醇(bME)/二硫代-b(ME)。为了增加重折叠效率,使用助溶剂例如丙三醇、多种分子量的聚乙二醇和精氨酸可能是必须的。
在一个实施方案中,本发明涉及线性载体,所述的线性载体包含来自核酸片段文库的核酸片段和编码N末端氨基酸缺失的报告蛋白的核苷酸序列。在另一个实施方案中,该线性载体还包含编码靶蛋白的核苷酸序列。
本发明还涉及多个用本发明的线性载体库转化的报告蛋白缺乏的宿主细胞。在一个实施方案中,该宿主细胞还用编码靶蛋白的核酸进一步转化。
下列实施例说明但不限制本发明的方法和组合物。其他的对在临床治疗中一般遇到的各种条件和参数的适当的修改和改进且它们是对本领域技术人员显而易见的也在本发明的精神和范围内。
实施例1转化酶缺乏的酵母突变体的制备
为了快速筛选无法生产的蛋白质的翻译融合配偶体(TFP),建立了一种自动筛选系统,它使用酵母转化酶作为报告蛋白来评价在蔗糖培养基中的细胞生长情况。
为了正确筛选出有用的TFP,需要不具有转化酶活性的酵母菌株使用转化酶基因作为报告基因。因此,野生型酵母的染色体SUC2基因被删除。为了制备SUC2缺失盒,质粒pRB58(Carlson等人,Cell 20:145(1982))用EcoRI和XhoI消化,并使SUC2编码基因恢复且将其引入pBluescript KS+(Stratagene,USA)的EcoRI-XhoI位点,从而生成pBIΔBX。如图1所示,在两个末端具有190bp重复序列(Tcl90)(Bae等人,Yeast 21:437(2004))的URA3基因插入含在pBIΔBX中的SUC2基因的HindIII-XbaI位点,从而生成pBIU。pBIU用EcoRI和XhoI消化,并根据醋酸锂方法(Hill等人,Nucleic Acids Res.19:5791(1991))将其转化至酿酒酵母菌Y2805(Mat a ura3SUC2pep4::HIS3 GALI canl)和Y2805Δgall(Mat a ura3 SUC2 pep4::HIS3 gal1 canl)株中(SK Rhee,Korea Research Instituteof Bioscience and Biotechnology)。转化细胞Y2805Δsuc2U(Mat a suc2::URA3 pep4::HIS3GALI canl)、Y2805Δgal1Δsuc2U(Mat a suc2::URA3 pep4::HIS3 gal1 canl)在缺乏尿嘧啶的选择培养基中被选择出来。
为了评价转化细胞的转化酶活性,分别在含有葡萄糖和蔗糖作为唯一碳源的两种培养基中培养单一菌落。结果,与对照相比菌落在葡萄糖培养基中正常生长但在蔗糖培养基中生长非常缓慢。为了研究分泌至培养基中的转化酶的数量,SUC2+株和Δsuc2株在YPD培养基(1%酵母提取物、2%细菌用蛋白胨和2%葡萄糖)中培养。培养液上清中含有的蛋白质用SDS-PAGE分离,并且将凝胶在蔗糖溶液中培养30分钟而后用染料TTC(2,3,5-三苯基-四氮唑盐酸盐)进行酶谱分析。如图2所示,发现Δsuc2株失去大部分转化酶活性。但是,该突变株具有在蔗糖培养基中以非常缓慢的速度生长的问题。相信这是由于线粒体功能的葡糖异生作用导致的细胞局部生长。因此为了解决这一问题,将抗菌素A,它是一种线粒体电子转移抑制剂,添加至培养基中以阻断细胞生长。结果,突变株的生长在含有抗菌素A的YSPA(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、1μg/ml抗菌素A和2%琼脂)或YPSGA(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、1μg/ml抗菌素A和2%琼脂)培养基中被完全抑制(图3)。
为了恢复选择株Y2805Δsuc2U(Mat a suc2::URA3 pep4::HIS3 GALI canl)和Y2805Δgal1Δsuc2U(Mat a suc2::URA3 pep4::HIS3 gal1 canl)的尿嘧啶营养缺陷,用含有TFP文库的URA3载体,需要除去用于删除SUC2基因的URA3基因。为此,将细胞在含有5-氟乳清酸(5-FOA)的培养基中培养且选择失去URA3基因的细胞,从而获得了URA3敲除菌株,Y2805Δsuc2(Mat a ura3 suc2::Tc190 pep4::HIS3 GALI canl)和Y2805Δgal1Δsuc2(Mat a ura3 suc2::Tc190pep4::HIS3 gal1 canl)(图1)。进行DNA印迹分析来证实染色体上的SUC2基因已删除,正如所预期的,并且URA3基因从整合的转座子中删除(敲除)(图4)。当酿酒酵母菌Y2805的染色体DNA用EcoRI处理和使用SUC2基因作为探针用DNA印迹分析时,检测到大约4.3kb的片段。当插入URA3基因(Y2805Δgal1Δsuc2U)时,该4.3kb的片段增加至大约5.0kb,而当敲除URA3基因(Y2805Δgal1Δsuc2U)时它减小至大约3.7kb。如图4所示,如预期的一样,显然SUC2基因被删除并失去了URA3基因(敲除)。
实施例2使用转化酶作为分泌报告蛋白的自动筛选系统的开发
使用两种人治疗蛋白质,即在酵母中良好分泌的人血清白蛋白(HSA)和酵母中几乎不分泌的人白细胞介素-2(IL-2),根据融合了转化酶的蛋白质的表达评价转化酶缺失株在蔗糖培养基中自动筛选的可能性。
构建三个质粒pYGAP-SNS-SUC2、pYGAP-HSA-SUC2和pYGAP-hIL2-SUC2来检测在蔗糖培养基中的自动选择。为了构建pYGAP-SUC2,其中pYGAP-SUC2含有在酵母GAPDH启动子控制下的转化酶基因(SUC2,YIL162W)表达盒,首先构建pST-SUC2,使用引物SUC-F(SEQ ID NO.1)和SUC-R(SEQ ID NO.2)扩增来自pBIΔBX(图1)的含有SUC2基因的PCR产物,将含有SUC2基因的PCR产物亚克隆至pST-Blue-1(Novagen,USA)中。用Pfu聚合酶(Stratagene,USA)或Ex-Taq DNA聚合酶(TaKaRa Korea BiomedicalInc.,Seoul,Korea)进行PCR。PCR条件包括一次94℃5分钟的循环和25次94℃30秒、55℃30秒和72℃2分钟的循环,随后最后循环72℃7分钟一次。将含有来自pST-SUC2的SUC2的EcoRI-SalI片段亚克隆至EcoRI-SalI消化的含有GAPDH启动子的YGAPα-HIR中以代替YEGα-HIR525的GAL10启动子(Sohn等人,Process Biochem.30:653(1995)),获得的质粒命名为pYGAP-SUC2。为了促进外源基因和SUC2的融合以及通过酵母二肽蛋白酶Kex2p诱导被融合蛋白的体内断裂(Mizuno K.等人,Biochem.Biophys.Res.Commun.156:246(1988)),在分泌中,将一个用于SfiI和NotI两个识别位点的人工合成序列和编码Kex2p断裂位点的序列(Leu-Asp-Lys-Arg(SEQ ID NO:214))通过PCR加入SUC2的分泌信号序列(19个氨基酸)和SUC2成熟序列(513个氨基酸)之间的结构框架内。两个PCR片段,即含有GAPDH启动子和SUC2分泌信号序列的PCR-A和含有SUC2成熟部分的PCR-B分别从pYGAP-SUC2中进行扩增,其中PCR-A使用引物GAP-F(SEQ ID NO:3)和SUCSSR(SEQ ID NO:4)扩增而PCR-B使用引物SUCM-F(SEQID NO:5)和SUC-R(SEQ ID NO:2)扩增。将这两个片段亚克隆至pST-Blue-1并通过SacI-NotI消化PCR-A和NotI-SalI消化PCR-B来恢复。酶消化的PCR-A和PCR-B共连接至SacI-SalI消化的pYGAP-SUC2上,获得的质粒命名为pYGAP-SNS-SUC2。为了构建质粒pYGAP-HSA-SUC2,该质粒在人血清白蛋白(HSA)和SUC2之间含有框架内融合基因,HAS基因使用引物HAS-F(SEQ ID NO:6)和HAS-R(SEQ ID NO:7)从pYHSA5中进行扩增(Kang等人,J.Microbiol.Biotechnol.8:42(1998))且在pST-Blue-1中亚克隆。含有HAS基因的SfiI消化的DNA亚克隆至SfiI消化的pYGAP-SNS-SUC2载体中。获得的质粒命名为pYGAP-HSA-SUC2。为了构建在人白细胞介素-2(hIL2)和SUC2间含有框架内融合基因的质粒pYGAP-hIL2-SUC2,使用引物IL2-F(SEQ ID NO:8)和IL2-R(SEQ IDNO:9)从pT7-hIL2(JK Jung,Korea Reasearch Institute of Bioscience and Biotechnology)中扩增hIL2基因且将其亚克隆至pST-Blue-1。然后,通过将SfiI消化的hIL2片段亚克隆至SfiI消化的pYGAP-SNS-SUC2载体中来构建质粒pYGAP-hIL2-SUC2。
表达人血清白蛋白和转化酶的融合蛋白的pYGAP-HSA-SUC2载体、表达IL-2和转化酶的融合蛋白的pYGAP-hIL2-SUC2和仅表达转化酶的pYGAP-SNS-SUC2分别转化至缺失内源性转化酶基因从而不能在蔗糖培养基中生长的酵母菌株(Y2805Δsuc2)中。将转化细胞涂布在含有葡萄糖作为唯一碳源的UD平板(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)和含有蔗糖作为唯一碳源的YPSA培养基(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、1μg/ml抗菌素A和2%琼脂)上且观察每个转化细胞的生长状况(图5)。当细胞用表达转化酶的pYGAP-SNS-SUC2转化时,它们在两种碳源中均正常生长。类似的,当细胞用HAS与转化酶的N末端融合的pYGAP-HSA-SUC2转化时,它们在两种碳源中均良好生长。相反,当细胞用IL2代替HSA进行融合的pYGAP-hIL2-SUC2转化时,它们在葡萄糖培养基中正常生长而在蔗糖培养基中几乎不生长。pYGAP-hIL2-SUC2转化细胞不能在蔗糖培养基中生长是由于IL-2无法由细胞分泌并导致与其融合的转化酶分泌的阻断。这些结果暗示使用转化酶作为任何来源的DNA的分泌信号和融合配偶体(翻译融合配偶体TFP)的报告蛋白的正向选择系统能使不或几乎不分泌的蛋白质例如人IL2的分泌提高。
实施例3构建翻译融合配偶体(TFP)文库的载体的制备
设计了数个载体用来从任何来源的基因组DNA或cDNA文库中构建TFP库。为了由cDNA构建TFP文库,构建了质粒YGaINV(图6)。使用两个PCR引物,即SfiI-SUC-F(SEQ IDNO:10)和SUC-Xho-R(SEQ IDNO:11)进行PCR来扩增来自pYGAP-hIL2-SUC2的编码转化酶的DNA片段。PCR条件包括一次94℃ 5分钟的循环和25次94℃ 30秒、55℃ 30秒和72℃ 2分钟的循环,最后循环72℃ 7分钟一次。然后EcoRI-SalI消化的PCR片段连接至EcoRI-SalI消化的YEGα-HIR525上,获得的质粒命名为YGaINV(图6)。为了从部分消化的基因组DNA中构建TFP文库,构建三个载体YGaF0INV、YGaF1INV和YGaF2INV,其中每个载体含有SUC2基因的三个不同阅读框中的一个(图7)。使用一个常用的正向引物Gal100-F(SEQ ID NO:12)和三个带有不同阅读框的反向引物Xho-F0-R(SEQ ID NO:13)、Xho-F1-R(SEQ ID NO:14)和Xho-F2-R(SEQ ID NO:15)以YGaINV作为模板进行三个不同的PCR扩增。使用Pfu聚合酶(Stratagene,USA)完成PCR。PCR条件包括一次94℃ 5分钟的循环和25次94℃ 30秒、55℃ 30秒和72℃ 2分钟的循环,最后循环72℃ 7分钟一次。三个PCR片段从琼脂糖凝胶上洗脱且用SfiI消化。然后将它们分别亚克隆至SfiI消化的YGaINV内。获得的三个质粒命名为YGaF0INV、YGaF1INV和YGaF2INV(图7)。
实施例4构建与酵母转化酶融合的cDNA文库
为了构建cDNA文库,将总的RNA从酿酒酵母菌Y2805(Mat aura3his3 pep4::HIS3canl)中分离出来。将酵母细胞在YPD培养基(2%酵母提取物、1%细菌用蛋白胨和2%葡萄样)中培养至对数生长期(mid-exponential phase)。将总RNA用Elion等人描述(Elion等人,Cell 39:633(1984))的方法分离出来。使用Oligotex mRNA试剂盒(Qiagen,Germany)从总RNA中纯化poly(A)+mRNA。cDNA使用SMART cDNA合成试剂盒(BDBioscience,USA)从分离的mRNA中合成出来。一个特别设计的引物ASA24N6(SEQ IDNO:16)代替SMART试剂盒中包括的引物用于合成cDNA的第一条链。由于引物ASA24N6设计含有一个SfiI识别位点和一个随机六核苷酸序列,它用于根据SMART试剂盒操作手册中描述的方法通过逆转录作用由mRNA合成cDNA的第一条链(图8)。由于引物ASA24N6是随机的六核苷酸序列,因此它可随机结合于mRNA的任何位置。因此,大多数使用此方法扩增的cDNA的第一条链含有编码酵母基因N末端部分的5’端部分序列。带有5’端部分序列的cDNA第一条链文库用作双链cDNA合成的PCR模板,使用SMART试剂盒(BD Bioscience,USA)5’端PCR引物和引物ASA24(SEQ IDNO:17)。获得的PCR产物包含大量的两端带有SfiI位点的cDNA的5’端部分片段。PCR条件包括如试剂盒推荐的一次95℃20秒的循环和20次95℃30秒、68℃6分钟的循环。扩增的cDNA用苯酚/氯仿/异戊醇(25∶24∶1)处理,并用2体积乙醇和0.1体积3M乙酸钠(pH 5.0)沉淀。获得的cDNA用SfiI在50℃消化2小时然后用琼脂糖凝胶电泳分离。0.5-1kbDNA使用凝胶提取试剂盒(Bioneer,Korea)从凝胶中分离。提取的DNA连接至SfiI消化的YGaINV载体上(图6)并转化至大肠杆菌DH5α中。转化的大肠杆菌在含有氨苄西林的LB培养基(1%细菌用蛋白胨、0.5%酵母提取物、1%NaCl和50μg/ml氨苄西林)上涂板且37℃培养过夜。大约5×104个大肠杆菌菌落混合至无菌蒸馏水中,使用质粒分离试剂盒(Bioneer,Korea)来分离总质粒,其中该总质粒中含有与SUC2基因融合的随机引物引导的cDNA文库。
实施例5构建与酵母转化酶融合的基因组DNA文库
实施例4中构建的TFP文库是从cDNA文库中获得的,其中该cDNA文库是从mRNA池中合成的。由于与几乎不表达的基因的mRNA相比,高表达基因的mRNA通常很充足,因此,TFP文库偏向于来自高表达基因。此外,一些基因在生长时期中的某一点被完全抑制从而不能在TFP文库中扩增,即使它们是TFP的良好候选者。为了解决这些问题,基因组DNA还用于构建TFP文库。如图9中所示,酿酒酵母菌Y2805的基因组DNA用Sau3AI部分消化然后在70℃培养10分钟使酶失活。在25℃,1小时,用Klernow片段和0.2mM dTTP和dCTP补平DNA形成二碱基的互补端,然后将0.5-1kb的DNA从琼脂糖凝胶中分离出来。此外,载体YGaF0INV、YGaF11NV、YGaF2INV(图7)用XhoI消化。70℃灭活酶10分钟后,用Klenow片段和0.2mM dTTP和dCTP补平载体形成二碱基互补端,然后从琼脂糖凝胶中纯化。每个载体用部分消化的基因组DNA连接然后分别转化至大肠杆菌DH5α。转化的大肠杆菌在含有氨苄西林的LB培养基(1%细菌用蛋白胨、0.5%酵母提取物、1%NaCl和50μg/ml氨苄西林)上涂板且37℃培养过夜。将从三个不同载体获得的大约2×105个大肠杆菌菌落倒入无菌蒸馏水中,使用质粒分离试剂盒(Bioneer,Korea)来分离含有与SUC2基因融合的基因组DNA文库的总质粒。
实施例6构建分泌转化酶的TFP文库
为了从实施例4和5中构建的基因组和cDNA文库中初选分泌转化酶的TFP文库,将DNA文库根据醋酸锂方法(Hill等人,Nucleic Acid Res.19:5791(1991))转化至酿酒酵母菌Y2805Δgal1Δsuc2(Mat a ura3 suc2::Tc190 pep4::HIS3 gal1 canl)。Y2805Δgal1Δsuc2不能使用蔗糖和半乳糖作为碳源由于缺失这两种基因。转化细胞涂布在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)和YPSGA培养基(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、0.3%半乳糖、1μg/ml抗菌素A和2%琼脂)上并在30℃培养4-6天。从cDNA和基因组DNA文库分别获得大约3000和1000个转化细胞。YPSGA培养基上生长的所有转化细胞用牙签转移至UD平板上且在30℃培养2天。使用玻璃珠从混合的细胞中分离出总DNA然后DNA用乙醇沉淀。为了得到含有TFP文库的质粒,将总DNA再转化至大肠杆菌DH5α中。转化的大肠杆菌在含有氨苄西林的LB培养基(1%细菌用蛋白胨、0.5%酵母提取物、1%NaCl和50μg/ml氨苄西林)上涂板且37℃培养过夜。大约获得2×104个大肠杆菌转化细胞并用无菌蒸馏水收集,使用质粒分离试剂盒(Bioneer,Korea)来分离总质粒。从而构建含有高达4000个单独诱导转化酶分泌的TFPs的TFP文库。从该文库中随机选择的质粒的核苷酸序列显示所有TFPs来源于单独编码不同分泌蛋白的酵母基因。
实施例7通过体内重组构建适用于许多靶蛋白的TFP文库载体
实施例6中收集了大约4000个具有分泌转化酶可能性的TFPs。为了开发方便适用于任何靶基因的TFP文库载体,设计出一个简单的体内重组系统。首先构建载体YGadV45(图10),通过体内重组用于将任何靶蛋白基因框内插入TFP文库和SUC2基因间。YGadV45包含缺陷型的SUC2(dSUC2),即N末端45个氨基酸缺失的SUC2,因此没有转化酶活性。此载体还设计在dSUC2前含有一个NotI和两个SfiI识别序列,一个作为重组靶点的连接序列和一个SwaI识别序列来通过体内重组简单插入TFP库和靶基因。使用正向引物INV45-F(SEQ ID NO:18)和反向引物SUC-Xho-R(SEQ ID NO:11)和Pfu聚合酶(Stratagene,USA)根据模板YGaINV来进行PCR。PCR条件包括一次94℃ 3分钟的循环和25次94℃ 30秒、55℃ 30秒和72℃ 90秒的循环,最后循环72℃ 7分钟一次。N末端修饰的缺陷型SUC2的基因片段从PCR中获得。NotI-SalI消化的PCR片段亚克隆至NotI-SalI消化的载体YGaINV上(图6),获得的质粒命名为YGadV45(图10)。为了构建YGadV45中的TFP文库,实施例6中得到的TFP文库用SfiI消化且在琼脂糖凝胶中分离。使用凝胶提取试剂盒(Bioneer,Korea)从凝胶中分离出大约0.5至1kb的DNA片段。纯化的DNA亚克隆至SfiI消化的YGadV45上(图10)且转化到大肠杆菌DH5α中。转化的大肠杆菌在含有氨苄西林的LB培养基(1%细菌用蛋白胨、0.5%酵母提取物、1%NaCl和50μg/ml氨苄西林)上涂板且37℃培养过夜。大约5×104个大肠杆菌转化细胞用无菌蒸馏水收集来分离总质粒。总质粒使用质粒分离试剂盒(Bioneer,Korea)来分离。分离的载体含有选自实施例6的与缺陷型的SUC2融合的TFPs。因此将此TFP文库载体转化至酿酒酵母菌Y2805Δgal1Δsuc2中(Mat a ura3 suc2::Tc190pep4::HIS3 gal1 canl)在UD平板(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)上获得数千个转化细胞而在YPSGA培养基(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、0.3%半乳糖、1μg/ml抗菌素A和2%琼脂)上没有转化细胞。因此,它在YPSGA培养基上可以极大降低选择的背景水平。仅具有包含TFP和SUC2间框内插入靶基因的载体的细胞可以在正确体内重组后在YPSGA上生长。TPF文库载体包含分别用于线性化和同源重组的一个罕见切割限制性酶SwaI的位点和一个TFP文库和dSUC2间的连接序列。
实施例8从TFP文库中自动选择分泌靶蛋白的最佳TFP
为了通过体内重组将靶蛋白框内融合至实施例7中开发的TFP文库的载体内,靶基因必须在5’末端具有连接DNA和3’末端具有SUC2的N末端部分。为了将此序列添加至靶基因的末端,使用重叠延伸PCR。使用靶点特异的正向引物KR-target-F(SEQ IDNO:19)和靶点特异的反向引物Target-INV-R(SEQ ID NO:20)进行第一步PCR,从包含靶基因的质粒中扩增编码成熟蛋白质的靶基因。还使用正向引物KR-Inv-F(SEQ ID NO:21)和反向引物Inv500-R(SEQ ID NO:22)单独进行另一PCR,从YGaINV(图6)中扩增将与靶基因的3’末端融合的SUC2的N末端部分。用Pfu聚合酶(Stratagene,USA)进行PCR,PCR条件包括一次94℃3分钟的循环和25次94℃30秒、55℃30秒和72℃90秒的循环,最后循环72℃7分钟一次。然后使用正向引物LNK40(SEQ ID NO:23)和反向引物Inv500-R(SEQ ID NO:22)从第一步中扩增的两个DNA片段中进行第二步PCR。获得的片段(插入片段)分别在5’末端包含40个核苷酸的连接DNA和在3’末端包含编码Kex2p识别位点(Leu-Asp-Lys-Arg(SEQ ID NO:214))的500bp的DNA和转化酶的N末端部分。为了进行体内重组,插入片段与实施例7中构建的SwaI消化的TFP文库载体以2∶1的比例混合且用于转化至酿酒酵母菌Y2805Δgal1Δsuc2中(Mat a ura3 suc2::Tc190pep4::HIS3 gal1 canl)(图11)。转化细胞涂布在YPSGA培养基(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、0.3%半乳糖、1μg/ml抗菌素A和2%琼脂)上且培养5天。仅有插入片段和包含适合TFP的载体通过体内重组的框内融合可以支持细胞生长在YPSGA培养基上。因此,使用这种方法,可通过在YPSGA培养基上简单选择生长细胞来获得用于任何靶蛋白的最佳TFP。
实施例9使用脂肪酶和转化酶的二重报告蛋白系统自动选择最佳TFP
使用如实施例8中描述的转化酶报告基因的自动选择系统对筛选用于靶蛋白例如IL2的最佳TFP十分有效,其中在实施例2中发现IL2能完全阻断转化酶的分泌。由于许多菌落可在蔗糖培养基上生长,因此从TFP文库中选择最佳TFP十分容易。然而,一些靶蛋白的融合即使连接弱TFP也不能完全阻断转化酶的分泌。这些遗漏的菌落也可在蔗糖培养基上生长。因此大量菌落应当检测其分泌水平来选择最佳TFP。为了解决这个费时的问题,开发了一种简单的选择方法,即使用成环报告蛋白脂肪酶在含有三丁酸甘油酯的平板上来鉴定具有高蛋白分泌水平的菌落。编码脂肪酶(CalB,南极假丝酵母的脂肪酶B)的基因框内融合至转化酶的5’末端。使用此二重报告蛋白系统,可在含有三丁酸甘油酯的YPSGA培养基中用转化酶和脂肪酶活性同时选择转化细胞。高水平分泌蛋白质的菌落可用菌落周围形成的环的大小来简单地确定。如图12所示,二重报告基因的构建通过三个PCR步骤来完成。含有CalB的1kb的PCR片段首先使用CalB正向引物KR-CalB-F(SEQ ID NO:24)和反向引物CalB-Inv-R(SEQ ID NO:25)从包含突变CalB基因的质粒pLGK-Lip14*(SY Kim,Ph.D.thesis,Yonsei University,Korea,2001)中进行扩增。含有SUC2基因5’端部分的0.5kb的PCR片段使用正向引物KR-Inv-F(SEQ ID NO:21)和反向引物Inv500-R(SEQ ID NO:22)单独从YGaINV中进行扩增(图6)。用Pfu聚合酶(Stratagene,USA)进行PCR,PCR条件包括一次94℃3分钟的循环和25次94℃ 30秒、55℃ 30秒和72℃ 90秒的循环,最后循环72℃ 7分钟一次。然后使用正向引物KR-CalB-F(SEQ ID NO:24)和反向引物Inv500-R(SEQ ID NO:22)从第一步中扩增的两个DNA片段中进行第二步PCR。靶基因的PCR使用正向引物KR-Target-F(SEQ ID NO:19)和Target-CalB-R(SEQ ID NO:26)从含有如实施例8中描述的靶基因的质粒中单独进行扩增。使用正向引物LNK40(SEQ ID NO:23)和反向引物Inv500-R(SEQ ID NO:22)从靶基因和融合了部分SUC2基因的CalB的混合物模板中进行第三步PCR。获得的DNA片段(插入片段)按顺序由40个核苷酸的连接子、靶基因、Kex2p断裂位点(Leu-Asp-Lys-Arg(SEQID NO:214))、CalB、Kex2p断裂位点(Leu-Asp-Lys-Arg(SEQ ID NO:214))和500bp的SUC2基因的5’端部分组成。为了进行体内重组,PCR扩增的插入片段与实施例7中构建的SwaI消化的TFP文库载体以2∶1的比例混合且用于转化至酿酒酵母菌Y2805Δgal1Δsuc2(Mat a ura3 suc2::Tc190 pep4::HIS3 gal1 canl)中。转化细胞分别涂布在YPSGA培养基(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、0.3%半乳糖、1μg/ml抗菌素A和2%琼脂)上用转化酶活性来选择和涂布在YPSGAT培养基(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、0.3%半乳糖、1μg/ml抗菌素A、1%三丁酸甘油酯和2%琼脂)上用转化酶和脂肪酶活性来选择。转化平板在30℃培养5天。分泌靶蛋白、脂肪酶和转化酶的菌落在YPSGA和YPSGAT平板上均可形成。如预期一样,菌落周围形成不同大小的环。环的大小与分泌的脂肪酶活性大概成正比。因此,从转化平板中很容易直接选择出高分泌水平的菌落(图13)。
实施例10选自TFP文库分泌人白细胞介素-2的新TFP
为了使用本发明中开发的方法鉴定最佳的TFPs,作为一种实例对一种几乎不能分泌的蛋白质人白细胞介素-2(hIL2)进行了试验。包含人IL2基因和500bp的SUC2基因的N末端部分的插入片段使用如实施例8所描述的PCR来扩增(图11)。使用正向引物KR-IL2-F(SEQ ID NO:27)和反向引物IL2-INV-R(SEQ ID NO:28)以pT7-hIL-2(JK Jung,Korea Research Institute of Bioscience and Biotechnology)作为模板进行PCR。为了扩增与IL2基因的3’末端融合的SUC2的N末端部分,使用正向引物KR-Inv-F(SEQ ID NO:21)和反向引物Inv500-R(SEQ ID NO:22)从YGaINV中(图6)单独进行了另一PCR。然后使用正向引物LNK40(SEQ ID NO:23)和Inv500-R(SEQ ID NO:22)对第一步中扩增的两个DNA片段来进行第二步PCR。获得的片段(插入片段)按顺序包含一个含有Kex2p识别序列(Leu-Asp-Lys-Arg(SEQ ID NO:214))的40个核苷酸的连接DNA、IL2、另一个Kex2p识别序列和转化酶的N末端部分。此片段与实施例7构建的SwaI消化的TFP文库载体共转化至酿酒酵母菌Y2805Δgal1Δsuc2(Mat aura3 suc2::Tc190 pep4::HIS3 gal1 can1)中。转化细胞涂布在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)和YPSGA培养基(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、0.3%半乳糖、1μg/ml抗菌素A和2%琼脂)上且在30℃培养5天。大约2×104个转化细胞从UD平板上获得而大约100个转化细胞从YPSGA上获得。三十个随机选择的生长在YPSGA上的转化细胞在YPD肉汤培养基中培养。分离出总的DNA且再将其转化至大肠杆菌DH5α中。转化的大肠杆菌在含有氨苄西林的LB培养基(1%细菌用蛋白胨、0.5%酵母提取物、1%NaCl和50μg/ml氨苄西林)上涂板且在37℃培养过夜。使用质粒提取试剂盒(Bioneer,Korea)从每个大肠杆菌转化细胞中分离出质粒。为了分析每个TFP的序列,一个结合于GAL10启动子的测序引物GAL100-F(SEQ ID NO:12)用于所有含有TFPs的质粒。核苷酸序列由Genotech Co.(Taejon,Korea)使用自动化测序设备(ABI Prism 377;PEBiosystems,Foster City,CA,USA)来确定。序列通过酵母菌基因组数据库(www.yeastgenome.org)的BLAST搜索来分析。结果,从生长在YPSGA培养基中的30个菌落中分离出的质粒中鉴定出九个新TFPs和一个已知TFP(TFP-3)(WO 2005/068658)。被分离的质粒分别命名为pYHTS-TFP9、pYHTS-TFP13、pYHTS-TFP17、pYHTS-TFP18、pYHTS-TFP 19、pYHTS-TFP20、pYHTS-TFP21、pYHTS-TFP25和pYHTS-TFP27。这九个新TFPs概述于表1中。
表1选择分泌人白细胞介素-2的TFPs
TFP编号 | 酵母开放阅读框架(ORF) | 融合氨基酸的数量(总) | 信号序列 | 蛋白质SEQ ID | DNASEQ ID |
TFP-9TFP-13TFP-17TFP-18TFP-19TFP-20TFP-21TFP-25TFP-27 | YGR106CYIL123WYNL190WYBR078WYJL178CYMR307WYOR247WYOR085WYKR042W | 217(265)127(350)68(204)199(467)144(271)187(559)55(210)190(350)89(450) | 前(24aa)前(19aa)前(20aa)前(20aa)前(19aa)前(22aa)前(19aa)前(17aa) | 293133353739414345 | 303234363840424446 |
实施例11使用选择的TFPs分泌人IL2
为了证实使用选择的TFPs能分泌人IL2,使用PCR构建了9个质粒以除去每个TFP的5’-UTR和实施例10中选择的质粒的SUC2(图14)。九个正向引物BamH-YGR-F(SEQID NO:47)、BamH-SIM-F(SEQ ID NO:48)、BamH-YNL-F(SEQ ID NO:49)、BamH-ECM-F(SEQ ID NO:50)、BamH-ATG-F(SEQ ID NO:51)、BamH-GAS-F(SEQ IDNO:52)、BamH-YOR-F(SEQ ID NO:53)、BamH-OST-F(SEQ ID NO:54)、BamH-UTH-F(SEQ ID NO:55)和一个常用反向引物IL2-TGA-R(SEQ ID NO:56)用于分别从质粒pYHTS-TFP9、pYHTS-TFP13、pYHTS-TFP17、pYHTS-TFP18、pYHTS-TFP19、pYHTS-TFP20、pYHTS-TFP21、pYHTS-TFP25和pYHTS-TFP27中进行PCR。九个PCR扩增片段用BamHI和SalI消化且从琼脂糖凝胶中分离出每一个。使用正向引物Sac-GAL-F(SEQ ID NO:57)和反向引物GAL-BamH-R(SEQ ID NO:58)从YEGα-HIR525中单独进行另一个PCR以扩增GAL启动子(Sohn等人,Process Biochem.30:653(1995))。SacI-BamHI消化的GAL启动子和九个BamHI-SacI消化的片段共连接至SacI-SalI消化的YEGα-HIR525上。获得的质粒分别命名为pYGT9-IL2(图15A)、pYGT13-IL2(图15B)、pYGT17-IL2(图15C)、pYGT18-IL2(图16A)、pYGT19-IL2(图16B)、pYGT20-IL2(图16C)、pYGT21-IL2(图17A)、pYGT25-IL2(图17B)和pYGT27-IL2(图17C)。人IL2表达载体pYGT9-IL2(大肠杆菌DH5α/pYGT9-IL2,图15A)和pYGT17-IL2(大肠杆菌DH5α/pYGT17-IL2,图15C)于2005年7月21日存放于国际保藏机构KCTC(韩国典型培养物保藏中心;韩国,大田,宇松区,欧洞52),登记号分别是KCTC 10828BP和KCTC10829BP。所有构建的载体的核苷酸序列证实具有正确的TFP和IL2间的框内融合且每个载体转化至酿酒酵母菌Y2805(Mat a ura3SUC2pep4::HIS3GAL1canl)中。转化细胞在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)上涂板且在30℃培养3天。每个转化的一个单独菌落接种至YPDG肉汤培养基(1%酵母提取物、2%细菌用蛋白胨、1%葡萄糖和1%半乳糖)且在30℃培养40小时。培养液上清(0.6ml)与冷丙酮混合至丙酮终浓度40%。在-20℃培养2小时后,蛋白质通过10000×g离心15分钟沉淀。冷冻干燥沉淀且重悬于1×SDS-PAGE样品缓冲液(Bio-Rad,USA)中用12%SDS-PAGE分析。凝胶染色剂(PhastGelBlue R,Pharmacia Biotech,USA)染色凝胶。如图18所示,分泌的IL2水平在TFPs间非常不同但都可以将人IL2分泌至培养液上清中。含有TFP 1-人IL2基因的质粒pYIL-KRT1-4(WO 2005/068658)用作对照。发现TFP9、13、21和27对人IL2的分泌十分有效(图18)。
实施例12选自TFP文库分泌人白细胞介素-32的新TFP
为了使用本发明中开发的方法鉴定最佳的TFPs,作为一种实例对一种很少分泌的蛋白质即新的人细胞因子,白细胞介素-32α(hIL32)(Kim等人,Immunity 22:131(2005))进行了试验。含有人IL32α基因和500bp的SUC2基因N末端部分的插入片段使用如实施例8所描述的PCR来扩增(图11)。使用正向引物KR-IL32α-F(SEQ ID NO:59)和反向引物IL32α-INV-R(SEQ ID NO:60)以pProExHTa-IL32α(DY Yoon,Konkuk University,Korea)作为模板进行PCR。为了扩增与IL32α基因的3’末端融合的SUC2的N末端部分,使用正向引物KR-Inv-F(SEQ ID NO:21)和反向引物Inv500-R(SEQ ID NO:22)从YGaINV(图6)中单独进行了另一PCR。然后使用正向引物LNK40(SEQ ID NO:23)和反向引物Inv500-R(SEQ ID NO:22)对从第一步中扩增的两个DNA片段进行第二步PCR。获得的片段(插入片段)按顺序包含一个含有Kex2p识别序列(Leu-Asp-Lys-Arg(SEQ IDNO:214))的40个核苷酸的连接DNA、IL32α、另一个Kex2p识别序列和转化酶的N末端部分。将此片段与实施例7构建的SwaI消化的TFP文库载体共转化至酿酒酵母菌Y2805Δgal1Δsuc2(Mat a ura3 suc2::Tc190pep4::HIS3gal1canl)中。转化细胞涂布在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)和YPSGA培养基(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、0.3%半乳糖、1μg/ml抗菌素A和2%琼脂)上且在30℃培养5天。大约2×104个转化细胞从UD平板中获得而大约250个转化细胞从YPSGA中获得。随机选择三十八个转化细胞在YPDG肉汤培养基(1%酵母提取物、2%细菌用蛋白胨、1%葡萄糖和1%半乳糖)中30℃培养40小时。培养液上清(0.6ml)与冷丙酮混合至丙酮终浓度40%。在-20℃培养2小时后,蛋白质通过10000×g离心15分钟沉淀。冷冻干燥沉淀且重悬于1×SDS-PAGE样品缓冲液(Bio-Rad,USA)中并用12%SDS-PAGE分析(图19)。根据大约20kDa处出现的蛋白质条带判断大多数转化细胞可以分泌人IL32α。其中,对显示深色IL32α条带的17个转化细胞进一步进行分析。每个转化细胞在YPD肉汤培养基中培养,分离出总DNA且将其再转化至大肠杆菌DH5α中。将转化的大肠杆菌在含有氨苄西林的LB培养基(1%细菌用蛋白胨、0.5%酵母提取物、1%NaCl和50μg/ml氨苄西林)上涂板且在37℃培养过夜。使用质粒提取试剂盒(Bioneer,Korea)从每个大肠杆菌转化细胞中分离出质粒。为了分析每个质粒的序列,一个结合于GAL10启动子的测序引物GAL100-F(SEQ ID NO:12)用于所有含有TFPs的质粒。核苷酸序列由Genotech Co.(Ta ejon,Korea)使用自动化测序设备(ABI Prism 377;PEBiosystems,Foster City,CA,USA)来确定。序列通过酵母菌基因组数据库(www.yeastgenome.org)的BLAST搜索来分析。结果从17个被选择的酵母菌株中分离出的质粒中鉴定出九个不同的TFPs。分离的质粒命名为pYHTS-IL32-TFP3、pYHTS-IL32-TFP11、pYHTS-IL32-TFP13、pYHTS-IL32-TFP21、pYHTS-IL32-TFP22、pYHTS-IL32-TFP25、pYHTS-IL32-TFP29、pYHTS-IL32-TFP34和pYHTS-IL32-TFP38。其中获得的TFP3、TFP13、TFP21和TFP25通常作为用于人IL2的最佳TFPs(WO2005/068658)且出现在实施例10中(表1)。五个分离IL32α的新TFPs概述于表2中。
表2分泌人白细胞介素-32α的新TFPs
TFP编号 | 酵母开放阅读框架(ORF) | 融合氨基酸的数量(总) | 信号序列 | 蛋白质SEQ ID | DNASEQ ID |
TFP-11TFP-22TFP-29TFP-34TFP-38 | YDR077WYJL159WYEL060CYLR390W-AYMR251W-A | 187(338)165(310)48(635)208(238)38(59) | 前(18aa)前原(19+54aa)前(19aa)前(22aa)前(20aa) | 6163656769 | 6264666870 |
实施例13使用选择的TFPs分泌人IL32α
为了证实使用选择的TFPs能分泌人IL32α,使用PCR构建数个质粒以除去每个TFP的5’-UTR和实施例12中选择的质粒的SUC2。六个正向引物BamH-CIS-F(SEQ IDNO:71)、BamH-SED-F(SEQ ID NO:72)、BamH-SIM-F(SEQ ID NO:73)、BamH-YOR247W-F(SEQ ID NO:74)、BamH-HSP-F(SEQ ID NO:75)、BamH-OST-F(SEQID NO:76)和一个常用反向引物IL32-TGA-R(SEQ ID NO:77)用于分别从质粒pYHTS-IL32-TFP3、pYHTS-IL32-TFP 11、pYHTS-IL32-TFP 13、pYHTS-IL32-TFP21、pYHTS-IL32-TFP22和pYHTS-IL32-TFP25中进行PCR。六个PCR扩增的片段用BamHI和SalI消化且从琼脂糖凝胶中分离出每一个。另一扩增GAL启动子的PCR使用正向引物Sac-GAL-F(SEQ ID NO:57)和反向引物GAL-BamH-R(SEQ ID NO:58)从YEGα-HIR525中进行(Sohn等人,Process Biochem.30:653(1995))。SacI-BamHI消化的GAL启动子和六个BamHI-SacI消化的片段共连接至SacI-SalI消化的YEGα-HIR525上。获得的质粒分别命名为pYGT3-IL32α、pYGT11-IL32α、pYGT13-IL32α、pYGT21-IL32α、pYGT22-IL32α和pYGT25-IL32α。所有构建的载体的核苷酸序列证实具有正确的TFP和IL32α间的框内融合且每个载体转化至酿酒酵母菌Y2805(Mat a ura3 SUC2 pep4::HIS3GAL1 canl)。转化细胞在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)上涂板且在30℃培养3天。每个转化的一个单独菌落接种至YPDG肉汤培养基(1%酵母提取物、2%细菌用蛋白胨、1%葡萄糖和1%半乳糖)且在30℃培养40小时。培养液上清(0.6ml)与冷丙酮混合至丙酮终浓度40%。在-20℃培养2小时后,蛋白质通过10000×g离心15分钟沉淀。冷冻干燥沉淀且将其重悬于1×SDS-PAGE样品缓冲液(Bio-Rad,USA)中并用12%SDS-PAGE分析。凝胶染色剂(PhastGelBlue R,Pharmacia Biotech,USA)染色凝胶。分泌的IL32α使用hIL32α单克隆抗体通过蛋白质印迹法进一步分析。蛋白质在CAPS缓冲液(2.2g/L CAPS、MeOH 10%、NaOH调整至pH11)中以300mA 90分钟转移至PVDE膜(Millipore,USA)。然后用人IL32抗体检测蛋白质(DY Yoon,Konkkuk University,Korea)。膜在4℃下在含有5%脱脂奶的PBS(137mMNaCl、2.7mM KCl、10mM Na2HPO4、2mM KH2PO4、HCl调整至pH 7.4)中封闭过夜。膜用含有0.05%吐温-20的PBS冲洗3次然后用含有3%脱脂奶的PBS稀释的一级抗体在室温孵育1小时。然后冲洗3次膜且用含有3%脱脂奶的PBS稀释的抗鼠二级抗体(Sigma Chemical Co.,USA)在室温孵育1小时。膜如上冲洗且用Sigma FastNBT/BCIP(Sigma Chemical Co.,USA)显影。如图20所示,所有选择的TFPs可将人IL32α分泌至培养液上清中。发现其中的TFP3、13、21和22对分泌人IL32α最佳。
实施例14分批补料发酵以生产人IL32α
pYGT3-IL32α转化的重组酵母菌株在5-L广口发酵罐中通过分批加料培养来评价分泌人IL32α的生产力。200ml接种培养液在1L培养瓶中使用基本培养基(0.67%无氨基酸酵母氮源、0.5%酪蛋白氨基酸和2%葡萄糖)培养。当使用发酵培养基(4%酵母提取物、1%蛋白胨、2%葡萄糖)作为初始发酵培养基的培养液的OD600到达大约15时,根据细胞生长速率以不同加液速率补充补料分批培养基(15%酵母提取物、30%葡萄糖)。培养液OD600达到大约130后,根据细胞生长速率以不同加液速率额外补充半乳糖(30%半乳糖)。大约72小时培养期后,培养液OD600达到大约220(图21A)。在给定时间点收集15μl培养基且通过SDS-PAGE估计分泌的蛋白质(图21B)。通过使用BCA蛋白质检测试剂(Pierce,USA)和光密度计(GS700,Bio-Rad,USA)的蛋白质直接测量法确定发现超过300mg/L的hIL32α分泌至培养基中。
实施例15使用酵母基因组数据库BLAST搜索以序列为基础选择TPFs
为了从酵母基因组中基于序列选择TFPs,18个选择的TFPs(4个来自WO2005/068658、9个来自实施例10和5个来自实施例12)的前分泌信号的氨基酸序列用作酵母菌基因组数据库(www.yeastgenome.org)BLAST搜索的查询序列。在BLASTP搜索中使用低期望阈值(100或1000),鉴定出数百个具有超过70%同源性的开放阅读框架(ORFs)。其中选择接近N末端具有序列同源性的ORFs,然后进一步进行SignalP(www.cbs.dtu.dk/services/SignalP-2.0/)分析来选择具有分泌信号的ORFs。结果随机选择出18个ORFs作为TFP候选。通过搜索鉴定的18个选择的ORFs是YGR279C(SCW4,细胞壁蛋白质)、YLR037C(DAN2,细胞壁甘露糖蛋白)、YLR110C(CCW12,细胞壁蛋白质)、YOR383C(FIT3,细胞壁甘露糖蛋白)、YIL011W(TLR3,细胞壁甘露糖蛋白)、YHR214W(假定的膜蛋白质)、YNL160W(YGP1,与细胞壁相关的分泌糖蛋白)、YGR296C-A(可疑的开放读码框)、YOL154W(ZPS1,假定的GPI锚蛋白质)、YPL187W(MFα,交配信息素α因子)、YHR214W(假定的膜蛋白质)、YKR013W(PRY2,未知功能蛋白质)、YHR139C(SPS100,孢子壁成熟所需蛋白质)、YIL169C(假定的未知功能蛋白质)、YOL155C(未定义ORF)、YMR325W(PAU19,假定蛋白质)、YDR134W(假定蛋白质)和YLR300W(EXG1,细胞壁主要外-1,3-β-葡聚糖酶)。分别使用酿酒酵母菌Y2805(Mat aura3 SUC2 pep4::HIS3 GAL1 canl)的基因组DNA对每个ORF进行扩增,所使用的引物对为:YGR279C使用YGR279C-F(SEQ ID NO:92)和YGR279C-R(SEQ ID NO:93)、YLR037C使用YLR037C-F(SEQ ID NO:94)和YLR037C-R(SEQ ID NO:95)、YLR110C使用YLR110C-F(SEQ ID NO:96)和YLR110C-R(SEQ ID NO:97)、YOR383C使用YOR383C-F(SEQ ID NO:98)和YOR383C-R(SEQ ID NO:99)、YIL011W使用YIL011W-F(SEQ ID NO:100)和YIL011W-R(SEQ ID NO:101)、YHR214W使用YHR214W-F(SEQ ID NO:102)和YHR214W-R(SEQ ID NO:103、YNL160W使用YNL160W-F(SEQ ID NO:104)和YNL160W-R(SEQ ID NO:105)、YGR296C-A使用YGR296C-A-F(SEQ ID NO:106)和YGR296C-A-R(SEQ ID NO:107)、YOL154W使用YOL154W-F(SEQ ID NO:108)和YOL154W-R(SEQ ID NO:109)、YPL187W使用YPL187W-F(SEQ ID NO:110)和YPL187W-R(SEQ ID NO:111)、YHR214W使用YHR214W-F(SEQ ID NO:112)和YHR214W-R(SEQ ID NO:113)、YKR013W使用YKR013W-F(SEQ ID NO:114)和YKR013W-R(SEQ ID NO:115)、YHR139C使用YHR139C-F(SEQ ID NO:116)和YHR139C-R(SEQ ID NO:117)、YIL169C 使用YIL169C-F(SEQ ID NO:118)和YIL169C-R(SEQ ID NO:119)、YOL155C使用YOL155C-F(SEQ ID NO:120)和YOL155C-R(SEQ ID NO:121)、YMR325W使用YMR325W-F(SEQ ID NO:122)和YMR325W-R(SEQ ID NO:123)、YDR134W使用YDR134W-F(SEQ ID NO:124)和YDR134W-R(SEQ ID NO:125)和YLR300W使用YLR300W-F(SEQ ID NO:126)和YLR300W-R(SEQ ID NO:127)。用Pfu聚合酶(Strategene,USA)进行PCR且PCR条件包括一次94℃3分钟的循环和25次94℃30秒、55℃30秒和72℃2分钟的循环,最后循环72℃7分钟一次。每个扩增的PCR片段由Genotech Co.(Taejon,Korea)使用自动测序设备(ABI Prism 377;PE Biosystems,Foster City,CA,USA)通过核苷酸测序来证实。
为了从选择的18个ORFs中筛选TFPs,对18个PCR片段的混合物进行了单向缺失且用于在YGadV45中构建TFP文库(图24)。使用引物SfiA-F(SEQ ID NO:128)根据由18个ORFs组成的模板通过单向PCR获得单链模板。用ExTaq(Takara Korea,Korea)进行PCR且PCR条件包括一次94℃3分钟的循环和30次94℃30秒、55℃30秒和72℃2分钟的循环,最后72℃7分钟循环一次。含有单链DNA的PCR产物使用PCR纯化试剂盒(Bioneer,Korea)来纯化。然后使用大肠杆菌DNA聚合酶I(NEB,England)和随机六核苷酸引物ASA24N6(SEQ ID NO:16)来进行双链DNA的再生。含有20μl模板DNA、1μlASA24N6引物、3μl 10×大肠杆菌DNApolI缓冲液、5μl 2.5mM dNTP和1μl大肠杆菌DNA polI的反应混合物在37℃培养1小时。使用PCR纯化试剂盒(Bioneer,Korea)来柱纯化DNA,使用引物SfiA-F(SEQ ID NO:128)和ASA24(SEQ ID NO:17)来进行PCR扩增。扩增的DNA再次柱纯化、用SfiI消化和通过琼脂糖凝胶电泳分离。0.5-1.0kb的SfiI消化的DNA亚克隆至SfiI处理的含有缺陷型SUC2(dSUC2)的YGadV45中。连接的DNA转化至大肠杆菌DH5α中。转化的大肠杆菌在含有氨苄西林的LB培养基(1%细菌用蛋白胨、0.5%酵母提取物、1%NaCl和50μg/ml氨苄西林)上涂板且在37℃培养过夜。大约1×104个大肠杆菌菌落混合至无菌蒸馏水且在YGadV45中含有18个ORFs的单向缺失的DNA片段文库的总质粒通过使用质粒分离试剂盒(Bioneer,Korea)来分离。
为了从18个ORFs的单向缺失的DNA片段文库中筛选适合的TFPs,一个编码人白细胞介素-2(hIL2)的基因插入该文库和dSUC2之间。含有hIL2基因和500bp SUC2的N末端部分的插入片段使用如实施例8中描述的方法使用PCR进行扩增(图11)。此片段与含有18个ORFs的单向缺失的DNA片段文库的SwaI消化的载体共转染至酿酒酵母菌Y2805Δgal1Δsuc2(Mat a ura3suc2::Tc190pep4::HIS3gal1canl)中。转化细胞涂布在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)和YPSGA培养基(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、0.3%半乳糖、1μg/ml抗菌素A和2%琼脂)上且在30℃培养5天。从UD平板上获得大约2×104个转化细胞而在YPSGA中获得大约数百个转化细胞。随机选择29个生长在YPSGA上的转化细胞在YPDG肉汤培养基(1%酵母提取物、2%细菌用蛋白胨、1%葡萄糖和1%半乳糖)中培养且在30℃培养40小时。培养液上清(0.6ml)与冷丙酮混合至丙酮终浓度40%。在-20℃培养2小时后,蛋白质通过10000×g离心15分钟沉淀。冷冻干燥沉淀且重悬于1×SDS-PAGE样品缓冲液(Bio-Rad,USA)中并用12%SDS-PAGE分析(图25)。发现数个转化细胞将人IL-2分泌至培养液上清中。从每个分泌人IL-2的细胞中分离出总DNA且将其再转化至大肠杆菌DH5α中。使用质粒提取试剂盒(Bioneer,Korea)从每个大肠杆菌转化细胞中分离出质粒。为了分析每个TFP的序列,一个结合于GAL10启动子的测序引物GAL100-F(SEQ ID NO:12)用于所有含有TFPs的质粒。核苷酸序列由Genotech Co.(Taejon,Korea)使用自动化测序设备(ABI Prism 377;PE Biosystems,Foster City,CA,USA)来确定。这些序列通过酵母菌基因组数据库(www.yeastgenome.orgg)的BLAST搜索来分析。结果从分泌人IL-2的12个转化细胞中分离的质粒中鉴定出六个新TFPs。分离的质粒分别命名为pYIL-TFP39、pYIL-TFP41、pYIL-TFP43、pYIL-TFP44、pYIL-TFP52和pYIL-TFP54。这六个新TFPs概述于表3中。
表3基于序列选择的ORFs中分泌人IL-2的TFPs
TFP编号 | 酵母开放阅读框架(ORF) | 融合氨基酸的数量(总) | 信号序列 | 蛋白质SEQ ID | DNASEQ ID |
TFP-39TFP-43TFP-44TFP-48TFP-52TFP-54 | YGR279CYLR110CYOR383CYGR279CYNL160WYLR037C | 57(386)129(133)71(204)119(386)129(354)124(124) | 前(19aa)前(18aa)前(18aa)前(19aa)前(19aa)前(20aa) | 129131133135137139 | 130132134136138140 |
实施例16通过单向缺失改变核心TFPs
为了改变实施例10和11中使用IL-2和IL-32α选择的14个TFPs(核心TFPs)和先前WO 2005/068658中鉴定的3个TFPs的有效性,将17个基因组开放阅读框架(ORFs),即YAR066W用于TFP-1、YFR026C用于TFP-2、YJL158C用于TFP3、YGR106C用于TFP-9、YDR077W用于TFP-11、YIL123W用于TFP13、YNL190W用于TFP-17、YBR078W用于TFP18、YJL178C用于TFP-19、YMR307W用于TFP-20、YOR247W用于TFP-21、YJL159W用于TFP-22、YOR085W用于TFP-25、YKR042W用于TFP-27、YEL060C用于TFP29、YLR390W-A用于TFP-34和YMR251W-A用于TFP-38进行如实施例15中描述的PCR扩增和单向缺失。每个开放阅读框架从酿酒酵母菌Y2805(Mat a ura3SUC2pep4::HIS3GAL1 canl)基因组DNA中分别使用PCR引物对:即YAR066W-F(SEQID NO:141)和YAR066W-R(SEQ ID NO:142)用于YAR066W、YFR026C-F(SEQ ID NO:143)和YFR026C-R(SEQ ID NO:144)用于YFR026C、YJL158C-F(SEQ ID NO:145)和YJL158C-R(SEQ ID NO:146)用于YJL158C、YGR106C-F(SEQ ID NO:147)和YGR106C-R(SEQ ID NO:148)用于YGR106C、YDR077W-F(SEQ ID NO:149)和YDR077W-R(SEQ ID NO:150)用于YDR077W、YIL123W-F(SEQ ID NO:151)和YIL123W-R(SEQ ID NO:152)用于YIL123W、YNL190W-F(SEQ ID NO:153)和YNL190W-R(SEQ ID NO:154)用于YNL190W、YBR078W-F(SEQ ID NO:155)和YBR078W-R(SEQ ID NO:156)用于YBR078W、YJL178C-F(SEQ ID NO:157)和YJL178C-R(SEQ ID NO:158)用于YJL178C、YMR307W-F(SEQ ID NO:159)和YMR307W-R(SEQ ID NO:160)用于YMR307W、YOR247W-F(SEQ ID NO:161)和YOR247W-R(SEQ ID NO:162)用于YOR247W、YJL159W-F(SEQ ID NO:163)和YJL159W-R(SEQ ID NO:164)用于YJL159W、YOR085W-F(SEQ ID NO:165)和YOR085W-R(SEQ ID NO:166)用于YOR085W、YKR042W-F(SEQ ID NO:167)和YKR042W-R(SEQ ID NO:168)用于YKR042W、YEL060C-F(SEQ ID NO:169)和YEL060C-R(SEQ ID NO:170)用于YEL060C、YLR390W-A-F(SEQ ID NO:171)和YLR390W-A-R(SEQ ID NO:172)用于YLR390W-A、YMR251W-A-F(SEQ ID NO:173)和YMR251W-A-R(SEQ ID NO:174)用于YMR251W-A来进行扩增。用Pfu聚合酶(Strategene,USA)进行PCR且PCR条件包括一次94℃3分钟的循环和25次94℃ 30秒、55℃ 30秒和72℃ 2分钟的循环,最后72℃ 7分钟循环一次。每个扩增的PCR片段由Genotech Co.(Taejon,Korea)使用自动测序设备(ABI Prism 377;PE Biosystems,Foster City,CA,USA)通过核苷酸测序来证实。
为了从17个开放阅读框架中筛选出变化的TFPs,其中从这17个开放阅读框架中获得了17个核心TFPs,对17个PCR片段混合物进行单向缺失且将其用于在YGadV45中构建TFP文库(图24)。使用引物SfiA-F(SEQ IDNO:128)根据由17个ORFs组成的模板通过单向PCR获得单链模板。用ExTaq(Takara Korea,Korea)进行PCR且PCR条件包括一次94℃ 3分钟的循环和30次94℃ 30秒、55℃ 30秒和72℃ 2分钟的循环,最后72℃ 7分钟循环一次。含有单链DNA的PCR产物使用PCR纯化试剂盒(Bioneer,Korea)来纯化。然后使用大肠杆菌DNA聚合酶I(NEB,England)和随机六核苷酸引物ASA24N6(SEQ ID NO:16)来进行双链DNA的再生。含有20μl模板DNA、1μl ASA24N6引物、3μl 10×大肠杆菌DNA polI缓冲液、5μl 2.5mM dNTP和1μl大肠杆菌DNA polI的反应混合物在37℃培养1小时。使用PCR纯化试剂盒DNA(Bioneer,Korea)来柱纯化DNA,并使用引物SfiA-F(SEQ ID NO:128)和ASA24(SEQ ID NO:17)进行PCR扩增。扩增的DNA再次柱纯化、用SfiI消化和通过琼脂糖凝胶电泳分离。0.5-1.0kb的SfiI消化的DNA亚克隆至SfiI处理的含有缺陷型SUC2(dSUC2)的YGadV45。连接的DNA转化至大肠杆菌DH5α中。转化的大肠杆菌在含有氨苄西林的LB培养基(1%细菌用蛋白胨、0.5%酵母提取物、1%NaCl和50μg/ml氨苄西林)上涂板且在37℃培养过夜。大约1×104个大肠杆菌菌落混合至无菌蒸馏水且在YGadV45中含有17个ORFs的单向缺失的DNA片段文库的总质粒通过使用质粒分离试剂盒(Bioneer,Korea)来分离。将来自于核心TFPs的17个ORFs和实施例15中制备的18个ORFs的两个单向缺失的文库的DNAs组合用于进一步分析。
为了从35个ORFs的单向缺失的DNA片段文库中筛选适合的TFPs,一个编码人白细胞介素-2(hIL2)的基因插入该文库和dSUC2之间。含有人IL2基因和500bp SUC2的N末端部分的插入片段使用如实施例8中描述的方法进行PCR扩增(图11)。此片段与含有35个ORFs的单向缺失的DNA片段文库的SwaI消化的载体共转染至酿酒酵母菌Y2805Δgal1Δsuc2(Mat a ura3 suc2::Tc190 pep4::HIS3 gal1 canl)中。转化细胞涂布在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)和YPSGA培养基(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、0.3%半乳糖、1μg/ml抗菌素A和2%琼脂)上且在30℃培养5天。从UD平板上获得大约2×104个转化细胞而从YPSGA中获得大约数百个转化细胞。随机选择24个生长在YPSGA上的转化细胞在YPDG肉汤培养基(1%酵母提取物、2%细菌用蛋白胨、1%葡萄糖和1%半乳糖)中培养且在30℃培养40小时。培养液上清(0.6ml)与冷丙酮混合至丙酮终浓度40%。在-20℃培养2小时后,蛋白质通过10000×g离心15分钟沉淀。冷冻干燥沉淀且重悬于1×SDS-PAGE样品缓冲液(Bio-Rad,USA)中并用12%SDS-PAGE分析(图26)。大多数转化细胞可将人IL-2分泌至培养液上清中但是它们之间具有不同水平。从每个分泌人IL-2的转化细胞中分离出总DNA且再将其转化至大肠杆菌DH5α中。使用质粒提取试剂盒(Bioneer,Korea)从大肠杆菌中分离出质粒。为了分析每个TFP的序列,一个结合于GAL10启动子的测序引物GAL100-F(SEQ ID NO:12)用于所有含有TFPs的质粒。核苷酸序列由Genotech Co.(Taejon,Korea)使用自动化测序设备(ABI Prism 377;PEBiosystems,Foster City,CA,USA)来确定。这些序列通过酵母菌基因组数据库(www.yeastgenome.org)的BLAST搜索来分析。结果从分泌人IL-2的18个转化细胞中分离出的质粒中鉴定出六个新TFPs。分离的质粒分别命名为pYIL-TFP40、pYIL-TFP50、pYIL43-TFP51、pYIL-TFP57、pYIL-TFP58和pYIL-TFP59。这六个新TFPs概述于表4中。
表4基于序列选择的开放阅读框架(ORFs)中分泌人IL-2的TFPs
TFP编号 | 酵母开放阅读框架(ORF) | 融合氨基酸的数量(总) | 信号序列 | 蛋白质SEQ ID | DNASEQ ID |
TFP-40TFP-50TFP-51TFP-57TFP-58TFP-59 | YGR279CYOR247WYOR247WYOL155CYAR066WYOR085W | 99(386)85(210)116(210)114(967)199(203)55(350) | 前(19aa)前(19aa)前(19aa)前(23aa)前(23aa)前(17aa) | 175177179181183185 | 176178180182184186 |
实施例17在核心TFPs间交换前(pre)和原(pro)信号序列人工合成TFPs
迄今为止,来自于结合(mating)因子α(MFα)的酵母分泌信号已被广泛用于酵母中各种重组蛋白质的分泌(Romanos等人,Yeast 8:423(1992))。该分泌信号包含19个氨基酸的前信号和66个氨基酸的原信号。原信号的准确功能还不确定但是已知它对于一些蛋白质的正确折叠和分泌十分重要。这一事实还在酵母中的一些重组蛋白质分泌中进行了研究(Chaudhuri等人,Eur.J.Biochem.206:793(1992))。在本发明中,两个分泌信号TFP-3和TFP-22被鉴定为前-原(pre-pro)类型。为了扩大本发明中选择的TFPs的用途,设计了具有不同起源的前和原信号的人工TFPs。使用TFP-1、2、3和4的前信号和一个常用的结合因子α的原信号构建四个人工TFPs,并将获得的TFPs命名为TFP-5、6、7和8。为了使这4个不同的前信号和一个常用的原信号间进行融合,使用了重叠延伸PCR。
分别使用引物对T1-F(SEQ ID NO:187)和T1-R(SEQ ID NO:188)、T2-F(SEQ IDNO:189)和T2-R(SEQ ID NO:190)、T3-F(SEQ ID NO:191)和T3-R(SEQ ID NO:192)、T4-F(SEQ ID NO:193)和T4-R(SEQ ID NO:194)根据质粒pYIL-KRTFP1、2、3和4(WO2005/268658)进行第一步PCR扩增4个TFPs的4个不同的前信号。还使用引物MF-Pro-F(SEQ ID NO:195)和MF-R(SEQ ID NO:196)从质粒YEGα-HIR525中单独进行另一PCR以扩增大约190bp结合因子α的原信号。然后分别使用4个不同正向引物即T1-F(SEQ ID NO:187)、T2-F(SEQ ID NO:189)、T3-F(SEQ ID NO:191)和T4-F(SEQ IDNO:193)和一个常用反向引物MF-R(SEQ ID NO:196)对第一步中扩增的4组2个DNA片段、4个前信号和一个MFα原信号来进行第二步PCRs用于4个不同的前-原信号。为了比较每个人工前-原信号序列与结合因子α的效率,结合因子α的前-原信号还使用引物MF-Pre-F(SEQ ID NO:197)和MF-R(SEQ ID NO”196)从YEGα-HIR525中进行PCR扩增。
选择靶蛋白人胰岛素样生长因子(hIGF)检测前-信号序列。已报道结合因子α的原信号对在酵母中分泌人胰岛素样生长因子十分必要(Chaudhuri等人,Eur.J.Biochem.206:793(1992))。人IGF基因使用引物KR-IGF-F(SEQ ID NO:198)和IGF-R(SEQ IDNO:199)从人cDNA文库(ES Choi,Korea Research Institute of Bioscience andBiotechnology,Korea)进行第一步PCR扩增,然后使用LNK40(SEQ ID NO:23)和IGF-R(SEQ ID NO:199)进行第二步PCR。含有IGF的DNA片段与使用5个正向引物,即T1-F(SEQ ID NO:187)、T2-F(SEQ ID NO:189)、T3-F(SEQ ID NO:191)、T4-F(SEQ IDNO:193)、MF-Pre-F(SEQ ID NO:197)和一个常用反向引物IGF-R(SEQ ID NO:199)先前扩增的5个含有前-原信号的PCR片段融合。所有融合的PCR产物用SfiI和SalI消化然后将其亚克隆至SfiI-SalI消化的载体YGalINV(图6)中。获得的质粒分别命名为pYGa-T 1α-IGF、pYGa-T2α-IGF、pYGa-T3α-IGF、pYGa-T4α-IGF和pYGa-MFα-IGF。将这五个质粒转化至酿酒酵母菌Y2805(Mat a ura3pep4::HIS3gal1canl)中。转化细胞涂布在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)上。分离每个转化的单一菌落并在YPDG (1%酵母提取物、2%细菌用蛋白胨、1%葡萄糖和1%半乳糖)中30℃培养40小时。培养液上清(0.6ml)与冷丙酮混合至丙酮终浓度40%。在-20℃培养2小时后,蛋白质通过10000×g离心15分钟沉淀。冷冻干燥沉淀且将其重悬于1×SDS-PAGE样品缓冲液(Bio-Rad,USA)中并用12%SDS-PAGE分析。分泌的IGF使用hIGF抗体通过蛋白质印迹法进一步分析(图27)。所有检测的前-原分泌信号可将人IGF分泌至培养液上清中但是具有不同效率。在5个前-原信号中,发现T3α(TFP-3的前信号和MFα的原信号)和T4α(TFP-4的前信号和MFα的原信号)对分泌人IGF有效。四个人工TFPs和一个新TFP概述于表5中。
表5分泌人IGF的新TFPs
TFP编号 | 酵母开放阅读框架(ORF) | 融合氨基酸的数量(总) | 信号序列 | 蛋白质SEQ ID | DNASEQ ID |
TFP-5TFP-6TFP-7TFP-8TFP-32 | YAR066W/YPL187WYFR026C/YPL 187WYJL158C/YPL187WHpPRB1/YPL187WYPL187W | 8884868384 | 前原(23+65aa)前原(19+65aa)前原(21+65aa)前原(18+65aa)前原(19+65aa) | 200202204206208 | 201203205207209 |
实施例18通过体内重组构建适用于许多靶基因的被选择的TFP载体
本发明中选择的三十五个TFPs(核心TFPs)(来自WO 2005/068658的4个TFPs、实施例10和11中使用两个报告蛋白选择的14个TFPs、实施例15中BLAST搜索选择的ORFs中的6个TFPs、实施例16中编码预先选择的TFPs的单向缺失的ORFs中的6个TFPs、实施例17中人工设计的TFPs中的5个TFPs)可能对分泌其它蛋白质有效。为了使大量的靶基因能使用这些载体,核心TFP载体使用靶基因通过体内重组重建。为了构建质粒YGaSW,使用引物GAL100-F(SEQ ID NO:12)和H77-1-R(SEQ ID NO:78)从YGadV45(图10)中进行PCR扩增含有EcoRI、2 SfiI、NotI、含有一个Kex2p识别位点的连接DNA、SwaI和SalI位点的170bp片段。将EcoRI-SalI消化的PCR片段亚克隆至EcoRI-SalI消化的YGadV45上,获得的质粒命名为YGaSW。此质粒包含EcoRI、SfiI、NotI、SfiI的限制性酶切位点、一个40bp连接子和GAL10启动子和GAL7终止子之间的限制性酶切位点SwaI和SalI。凝胶纯化每个核心TFP且将其亚克隆至SfiI消化的YGaSW上,获得的35个质粒分别命名为YGaSW-TFP1、YGaSW-TFP2、YGaSW-TFP3、YGaSW-TFP4、YGaSW-TFP5、YGaSW-TFP6、YGaSW-TFP7、YGaSW-TFP8、YGaSW-TFP9、YGaSW-TFP11、YGaSW-TFP13、YGaSW-TFP17、YGaSW-TFP18、YGaSW-TFP19、YGaSW-TFP20、YGaSW-TFP21、YGaSW-TFP22、YGaSW-TFP25、YGaSW-TFP27、YGaSW-TFP29、YGaSW-TFP32、YGaSW-TFP34、YGaSW-TFP38、YGaSW-TFP39,YGaSW-TFP40,YGaSW-TFP43,YGaSW-TFP44,YGaSW-TFP48、YGaSW-TFP50、YGaSW-TFP51、YGaSW-TFP52、YGaSW-TFP54、YGaSW-TFP57、YGaSW-TFP58和YGaSW-TFP59。
实施例19用于人生长激素分泌而选择的核心TFPs的评价
检测本发明选择的核心TFPs对人生长激素(hGH)的分泌。使用引物hGH-F(SEQ IDNO:79)和hGH-R(SEQ ID NO:80)PCR扩增从人cDNA文库(ES Choi,Korea ResearchInstitute of Bioscience and Biotechnology,Korea)中人GH基因且将其亚克隆至pST-Blue1(Novagen,USA)。获得的质粒命名为pST-hGH。使用引物KR-hGH-F(SEQ ID NO:81)和hGH-Sal-R(SEQ ID NO:82)对pST-hGH进行第二步PCR。含有hGH基因的PCR产物用于使用引物LNK40(SEQ ID NO:23)和GT70-R(SEQ ID NO:83)的第三步PCR以加入与实施例18中构建的YGaS W-TFP载体相同的序列。扩增的PCR片段与SwaI消化的YGaSW-TFP载体以2∶1混合且通过体内重组转化至酿酒酵母菌Y2805(Mat a ura3 SUC2pep4::HIS3 gal1 canl)中。转化细胞在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)上涂板在30℃培养3天。将每个转化的单一菌落接种至YPDG肉汤培养基(1%酵母提取物、2%细菌用蛋白胨、1%葡萄糖和1%半乳糖)中且在30℃培养40小时。培养液上清(0.6ml)与冷丙酮混合至丙酮终浓度40%。在-20℃培养2小时后,蛋白质通过10000×g离心15分钟沉淀。冷冻干燥沉淀且将其重悬于1×SDS-PAGE样品缓冲液(Bio-Rad,USA)中并用12%SDS-PAGE分析。如图22所示,大多数TFPs可将人生长激素分泌至培养液上清中。其中具有pYGT21-hGH的菌株被检测到在补料发酵中的分泌水平。在所示时间点取样的10μl培养液上清用SDS-PAGE分析(图23)。大约500mg/l人生长激素分泌至培养液上清中。
实施例20用于分泌人胱天蛋白酶-1亚基P10而选择的核心TFPs的评价
检测本发明中选择的核心TFPs对人胱天蛋白酶-1亚基p10(hP10)的分泌。使用引物KR-hP10-F(SEQ ID NO:210)和hP10-Sal-R(SEQ ID NO:211)对人cDNA文库(ES Choi,Korea Research Institute of Bioscience and Biotechnology,Korea)中的人p 10基因进行PCR扩增。含有hP10基因的PCR产物用于使用引物LNK40(SEQ ID NO:23)和GT70-R(SEQID NO:83)的第二步PCR以加入与实施例18中构建的YGaSW-TFP载体相同的序列。扩增的PCR片段与SwaI消化的YGaSW-TFP载体以2∶1混合且通过体内重组转化至酿酒酵母菌Y2805(Mat a ura3SUC2pep4::HIS3gal1canl)中。转化细胞在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)上涂板并在30℃培养3天。每个转化的单一菌落接种至YPDG肉汤培养基(1%酵母提取物、2%细菌用蛋白胨、1%葡萄糖和1%半乳糖)中且在30℃培养40小时。培养液上清(0.6ml)与冷丙酮混合至丙酮终浓度40%。在-20℃培养2小时后,蛋白质通过10000×g离心15分钟沉淀。冷冻干燥沉淀且将其重悬于1×SDS-PAGE样品缓冲液(Bio-Rad,USA)中并用12%SDS-PAGE进行分析。如图28所示,仅含有前-原信号的4个人工TPFs可将hP10分泌至培养液上清中。正如在hIGF中发现的情况一样,原信号对在酵母中适当分泌人胱天蛋白酶-1亚基P10是必须的。
实施例21用于分泌人白细胞介素-32γ而选择的核心TFPs的评价
检测本发明中选择的核心TFPs对人白细胞介素-32γ(hIL32γ)的分泌。使用引物KR-hIL32g-F(SEQ ID NO:212)和hIL32g-S al-R(SEQ ID NO:213)对pGMT-IL32γ(DY Yoon,Konkuk University,Korea)中的编码人白细胞介素32剪接变体γ的基因进行PCR扩增。含有hIL32γ基因的PCR产物用于使用引物LNK40(SEQ ID NO:23)和GT70-R(SEQ IDNO:83)进行第二步PCR以加入与实施例18中构建的YGaSW-TFP载体相同的序列。扩增的PCR片段与SwaI消化的YGaSW-TFP载体以2∶1混合且通过体内重组将其转化至酿酒酵母菌Y2805(Mat a ura3SUC2pep4::HIS3gal1can1)中。转化细胞在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)上涂板并在30℃培养3天。将每个转化的单一菌落接种至YPDG肉汤培养基(1%酵母提取物、2%细菌用蛋白胨、1%葡萄糖和1%半乳糖)中且在30℃培养40小时。培养液上清(0.6ml)与冷丙酮混合至丙酮终浓度40%。在-20℃培养2小时后,蛋白质通过10000×g离心15分钟沉淀。冷冻干燥沉淀且将其重悬于1×SDS-PAGE样品缓冲液(Bio-Rad,USA)中并用12%SDS-PAGE进行分析。在所有检测的TFPs中,TFP3和TPF27鉴定为对分泌人IL-32γ有效(图29)。
实施例22从酿酒酵母中选择的巴斯德毕赤酵母的TFP文库
本发明的TFP选择方法还可应用于其它来源的基因组或cDNA问库。使用巴斯德毕赤酵母菌(P.pastoris)作为mRNA来源的实例。从巴斯德毕赤酵母菌GS115(Invitrogen,USA)中分离出总RNA来构建cDNA文库。酵母在YPD培养基(2%酵母提取物、1%细菌用蛋白胨和2%葡萄样)中培养至对数生长期指数期。总RNA用Elion等人描述(Elion等人,Cell 39:633(1984))的方法从巴斯德毕赤酵母中分离出来。使用Oligotex mRNA试剂盒(Qiagen,Germany)从总RNA中纯化poly(A)+mRNA。cDNA使用SMART cDNA合成试剂盒(BD Bioscience,USA)从分离的mRNA中合成出来。一个特别设计的引物ASA24N6(SEQ ID NO:16)代替SMART试剂盒中包括的引物用于合成cDNA的第一条链,如实施例4中描述(图8)。引物ASA24N6由于其随机六核苷酸序列可随机结合于mRNA的任何位置。因此大多数使用此方法扩增的cDNA的第一条链含有编码酵母基因N末端部分的5’端部分序列。带有5’端部分序列的cDNA第一条链文库用作合成双链cDNA的PCR模板,其中使用SMART试剂盒(BD Bioscience,USA)5’端PCR引物和引物ASA24(SEQ ID NO:17)。使用此方法制备的PCR产物包含大量两端带有SfiI位点的cDNA的5’端部分片段。PCR条件包括如试剂盒介绍的一次95℃20秒的循环和20次95℃30秒、68℃6分钟的循环。扩增的cDNA用苯酚/氯仿/异戊醇(25∶24∶1)处理,并用2体积乙醇和0.1体积3M乙酸钠(pH 5.0)沉淀。获得的cDNA用SfiI在50℃消化2小时然后用琼脂糖凝胶电泳分离。0.5-1kb的DNA使用凝胶提取试剂盒(Bioneer,Korea)从凝胶中分离出来。提取的DNA连接至SfiI消化的YGaINV载体上并转化至大肠杆菌DH5α中。转化的大肠杆菌在含有氨苄西林的LB培养基(1%细菌用蛋白胨、0.5%酵母提取物、1%NaCl和50μg/ml氨苄西林)上涂板且37℃培养过夜。大约4×104个大肠杆菌菌落混合至无菌蒸馏水中,并使用质粒分离试剂盒(Bioneer,Korea)来分离含有合成的cDNA文库的总质粒,其中该cDNA文库通过融合于SUC2基因的随机引物合成。为了从巴斯德毕赤酵母菌种中选择分泌转化酶的TFP文库,将DNA文库根据醋酸锂方法(Hill等人,Nucleic Acids Res.19:5791(1991))转化至酿酒酵母菌Y2805Δgal1Δsuc2(Mat a ura3suc2::Tc190 pep4::HIS3 gal1 canl)中。转化细胞涂布在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)和YPSGA培养基(1%酵母提取物、2%细菌用蛋白胨、2%蔗糖、0.3%半乳糖、1μg/ml抗菌素A和2%琼脂)上且在30℃培养4-6天。大约1000个转化细胞从巴斯德毕赤酵母的cDNA文库中获得。随机选择生长在YPSGA培养基上的五个不同转化细胞且使用玻璃珠从每个菌落的培养细胞中分离出总DNA。然后用乙醇沉淀DNA。分离的DNA再转化至大肠杆菌DH5α中。大肠杆菌在含有氨苄西林的LB培养基(1%细菌用蛋白胨、0.5%酵母提取物、1%NaCl和50μg/ml氨苄西林)上涂板且在37℃培养过夜。使用质粒提取试剂盒(Bioneer,Korea)从转化的大肠杆菌中分离出质粒。为了分析每个从巴斯德毕赤酵母cDNA中获得的TFP的序列,使用一个结合于GAL10启动子的测序引物GAL100-F(SEQ ID NO:12)。核苷酸序列由Genotech Co.(Taejon,Korea)使用自动化测序设备(ABI Prism 377;PE Biosystems,FosterCity,CA,USA)来确定。这些序列用美国国家生物技术信息中心(NCBI)序列数据库(www.ncbi.nlm.nih.gov)的BLAST搜索检测。结果从5个选择的菌落中分离的质粒中鉴定出四个不同的巴斯德毕赤酵母TFPs。分离的质粒命名为pYHTS-PpTEP1、pYHTS-PpTEP2、pYHTS-PpTEP3和pYHTS-PpTEP4。这四个分离自巴斯德毕赤酵母的TFPs概述于表6中。
表6从巴斯德毕赤酵母中分离的TFPs
TFP编号 | 同族物 | 融合氨基酸的数量(信号) | 信号序列 | 蛋白质SEQ ID | DNASEQ ID |
PpTFP-1PpTFP-2PpTFP-3PpTFP-4 | SUN家族SED1未知类粘蛋白 | 1019482127 | 前(21aa)前(17aa)前(20aa)前(18aa) | 84868890 | 85878991 |
实施例23使用人IL2评价来自巴斯德毕赤酵母的TFPs
使用人IL-2来检测表6中概述的四个巴斯德毕赤酵母TFPs在酿酒酵母菌中的分泌效率。对质粒pYHTS-PpTFP1、pYHTS-PpTFP2、pYHTS-PpTFP3和pYHTS-PpTFP4中的每个PpTEP进行PCR扩增,分别使用引物对PpTFP1-F(SEQ ID NO:227)和PpTFP1-R(SEQ ID NO:228)、PpTFP2-F(SEQ ID NO:229)和PpTFP2-R(SEQ ID NO:230)、PpTFP3-F(SEQ ID NO:231)和PpTFP3-R(SEQ ID NO:232)、PpTFP4-F(SEQ ID NO:233)和PpTFP4-R(SEQ ID NO:234)对。用SfiI消化凝胶纯化的PCR片段且亚克隆至SfiI消化的YGaSW载体(图10)上,获得的质粒分别命名为YGaSW-PpTFP1、YGaSW-PpTFP2、YGaSW-PpTFP3和YGaSW-PpTFP4。
被扩增的PCR片段含有人IL-2基因,其中人IL-2基因上含有与YGaSW-PpTFP载体相同的序列,将该被扩增的PCR片段与SwaI消化的YGaSW-PpTFP载体以2∶1混合且通过体内重组转化至酿酒酵母菌Y2805(Mat a ura3 SUC2pep4::HIS3gal1can1)中。转化细胞在UD培养基(0.67%无氨基酸酵母氮源、0.77g/l氨基酸混合物、2%葡萄糖和2%琼脂)上涂板在30℃培养3天。每个转化的单一菌落接种至YPDG肉汤培养基(1%酵母提取物、2%细菌用蛋白胨、1%葡萄糖和1%半乳糖)中且在30℃培养40小时。培养液上清(0.6ml)与冷丙酮混合至丙酮终浓度40%。在-20℃培养2小时后,蛋白质通过10000×g离心15分钟沉淀。冷冻干燥沉淀且重悬于1×SDS-PAGE样品缓冲液(Bio-Rad,USA)中并用12%SDS-PAGE进行分析。如图30所示,所有PpTFPs可将人白细胞介素-2分泌至培养液上清中,显示了这两种酵母间TFP的相容性。
根据本发明的完整描述,本领域技术人员应当理解在不影响本发明的范围或其任何实施方案的情况下,在广泛和同等范围的条件、成分和其它参数下可以进行同样的方案。本文所引用的所有专利、专利申请和出版物全部是全文引入本文作为参考。
序列表
<110>Korea Research Institute of Bioscience and Biotechnology
Sohn,Jung-Hoon
Choi,Eui-Sung
Bae,Jung-Hoon
Lee,Eung-Suck
Shin,Mi-Kyung
Yoon,Sung-Sook
Chun,Chang-Soo
<120>Library of Translational Fusion Partners for Producing
Recombinant Proteins and Translational Fusion Partners Screened
There from
<130>2472.002PC01/EKS/RAS
<160>233
<170>PatentIn version 3.3
<210>1
<211>29
<212>DNA
<213>Artificial sequence
<220>
<223>SUC-F
<400>1
gaattcaaaa atgcttttgc aagctttcc 29
<210>2
<211>30
<212>DNA
<213>Artificial sequence
<220>
<223>SUC-R
<400>2
gtcgacttac tattttactt cccttacttg 30
<210>3
<211>24
<212>DNA
<213>Artificial sequence
<220>
<223>GAP-F
<400>3
gagctcaagc ttaccagttc tcac 24
<210>4
<211>41
<212>DNA
<213>Artificial sequence
<220>
<223>SUCSS-R
<400>4
gcggccgcac ggccgtaatg gcctgcagat attttggctg c 41
<210>5
<211>47
<212>DNA
<213>Artificial sequence
<220>
<223>SUM-F
<400>5
gcggccgcct cggccctaga taaaaggtca atgacaaacg aaactag 47
<210>6<211>33
<212>DNA
<213>Artificial sequence
<220>
<223>HSA-F
<400>6
ggccattacg gccgtgatgc acacaagagt gag 33
<210>7
<211>33
<212>DNA
<213>Artificial sequence
<220>
<223>HSA-R
<400>7
ggccgaggcg gcctaagcct aaggcagctt gac 33
<210>8
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>IL2-F
<400>8
ggccattacg gccgtgcacc tacttcaagt tctac 35
<210>9
<211>33
<212>DNA
<213>Artificial sequence
<220>
<223>IL2-R
<400>9
ggccgaggcg gccagttagt gttgagatga tgc 33
<210>10
<211>58
<212>DNA
<213>Artificial sequence
<220>
<223>SfiI-SUC-F
<400>10
gaattcaaaa ggccattacg gccgcggccg cctcggccct agataaaagg tcaatgac 58
<210>11
<211>29
<212>DNA
<213>Artificial sequence
<220>
<223>SUC-Xho-R
<400>11
ggctcgagct attttacttc ccttacttg 29
<210>12
<211>26
<212>DNA
<213>Artificial sequence
<220>
<223>GAL100-F
<400>12
gatatgtata tggtggtaat gccatg 26
<210>13
<211>41
<212>DNA
<213>Artificial sequence
<220>
<223>Xho-F0-R
<400>13
ctagggccga ggcggccctc gagggccgta atggcctttt g 41
<210>14
<211>42
<212>DNA
<213>Artificial sequence
<220>
<223>Xho-F1-R
<400>14
ctagggccga ggcggccgct cgagggccgt aatggccttt tg 42
<210>15
<211>43
<212>DNA
<213>Artificial sequence
<220>
<223>Xho-F2-R
<400>15
ctagggccga ggcggccgtc tcgagggccg taatggcctt ttg 43
<210>16
<211>30
<212>DNA
<213>Artificial sequence
<220>
<223>ASA24N6
<220>
<221>misc_feature
<222>(25)..(30)
<223>n is a,c,g,or t
<400>16
gccagcagag gccgaggcgg ccagnnnnnn 30
<210>17
<211>24
<212>DNA
<213>Artificial sequence
<220>
<223>ASA24
<400>17
gccagcagag gccgaggcgg ccag 24
<210>18
<211>65
<212>DNA
<213>Artificial sequence
<220>
<223>INV45-F
<400>18
gcggccgcct cggcctctgc tggcctcgcc ttagataaaa gatttaaatg acaccgtatg 60
gggta 65
<210>19
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>KR-target-F
<220>
<221>misc_feature
<222>(19)..(35)
<223>n is a,c,g,or t
<400>19
ctcgccttag ataaaagann nnnnnnnnnn nnnnn 35
<210>20
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>Target-INV-R
<220>
<221>misc_feature
<222>(19)..(35)
<223>n is a,c,g,or t
<400>20
cattgaacgc ttgtccaann nnnnnnnnnn nnnnn 35
<210>21
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>KR-Inv-F
<400>21
ttggacaagc gttcaatgac aaacgaaact agcgatag 38
<210>22
<211>25
<212>DNA
<213>Artificial sequence
<220>
<223>Inv500-R
<400>22
tcataatcca tttttgagaa ggttc 25
<210>23
<211>40
<212>DNA
<213>Artificial sequence
<220>
<223>LNK40
<400>23
ggccgcctcg gcctctgctg gcctcgcctt agataaaaga 40
<210>24
<211>30
<212>DNA
<213>Artificial sequence
<220>
<223>KR-CalB-F
<400>24
ttggacaagc gtctaccttc cggttcggac 30
<210>25
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>CalB-Inv-R
<400>25
cattgaacgc ttgtccaagg gggtgacgat gccggagc 38
<210>26
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>Target-CalB-R
<220>
<221>misc_feature
<222>(19)..(35)
<223>n is a,c,g,or t
<400>26
aggtagacgc ttgtccaann nnnnnnnnnn nnnnn 35
<210>27
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>KR-IL2-F
<400>27
ctcgccttag ataaaagagc acctacttca agttctac 38
<210>28
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>IL2-INV-R
<400>28
cattgaacgc ttgtccaaag ttagtgttga gatgatgc 38
<210>29
<211>217
<212>PRT
<213>Artificial sequence
<220>
<223>TFP9-AA
<400>29
Met Val Phe Gly Gln Leu Tyr Ala Leu Phe Ile Phe Thr Leu Ser Cys
1 5 10 15
Cys Ile Ser Lys Thr Val Gln Ala Asp Ser Ser Lys Glu Ser Ser Ser
20 25 30
Phe Ile Ser Phe Asp Lys Glu Ser Asn Trp Asp Thr Ile Ser Thr Ile
35 40 45
Ser Ser Thr Ala Asp Val Ile Ser Ser Val Asp Ser Ala Ile Ala Val
50 55 60
Phe Glu Phe Asp Asn phe Ser Leu Leu Asp Asn Leu Met Ile Asp Glu
65 70 75 80
Glu Tyr pro phe phe Asn Arg Phe Phe Ala Asn Asp Val Ser Leu Thr
85 90 95
Val His Asp Asp Ser Pro Leu Asn Ile Ser Gln Ser Leu Ser Pro Ile
100 105 110
Met Glu Gln phe Thr Val Asp Glu Leu Pro Glu Ser Ala Ser Asp Leu
115 120 125
Leu Tyr Glu Tyr Ser Leu Asp Asp Lys Ser Ile Val Leu Phe Lys Phe
130 135 140
Thr Ser Asp Ala Tyr Asp Leu Lys Lys Leu Asp Glu Phe Ile Asp Ser
145 150 155 160
Cys Leu Ser Phe Leu Glu Asp Lys Ser Gly Asp Asn Leu Thr Val Val
165 170 175
Ile Asn Ser Leu Gly Trp Ala Phe Glu Asp Glu Asp Gly Asp Asp Glu
180 185 190
Tyr Ala Thr Glu Glu Thr Leu Ser His His Asp Asn Asn Lys Gly Lys
195 200 205
Glu Gly Asp Asp Leu Ala Ala Ser Ala
210 215
<210>30
<211>728
<212>DNA
<213>Artificial sequence
<220>
<223>TFP9-nt
<400>30
ggccattacg gccggggatt gataataacc actgctgtga ctatatataa taagaatcga 60
actgtaaagt taaagcaatg gtgttcggtc agctgtatgc ccttttcatc ttcacgttat 120
catgttgtat ttccaaaact gtgcaagcag attcatccaa ggaaagctct tcctttattt 180
cgttcgacaa agagagtaac tgggatacca tcagcactat atcttcaacg gcagatgtta 240
tatcatccgt tgacagtgct atcgctgttt ttgaatttga caatttctca ttattggaca 300
acttgatgat tgacgaagaa tacccattct tcaatagatt ctttgccaat gatgtcagtt 360
taactgttca tgacgattcg cctttgaaca tctctcaatc attatctccc attatggaac 420
aatttactgt ggatgaatta cctgaaagtg cctctgactt actatatgaa tactccttag 480
atgataaaag catcgttttg ttcaagttta cctcggatgc ctacgatttg aaaaaattag 540
atgaatttat tgattcttgc ttatcgtttt tggaggataa atctggcgac aatttgactg 600
tggttattaa ctctcttggt tgggcttttg aagatgaaga tggtgacgat gaatatgcaa 660
cagaagagac tttgagccat catgataaca acaagggtaa agaaggcgac gatctggccg 720
cctcggcc 728
<210>31
<211>127
<212>PRT
<213>Artificial sequence
<220>
<223>TFP13-aa
<400>31
Met Lys Phe Ser Thr Ala Val Thr Thr Leu Ile Ser Ser Gly Ala Ile
1 5 10 15
Val Ser Ala Leu Pro His Val Asp Val His Gln Glu Asp Ala His Gln
20 25 30
His Lys Arg Ala Val Ala Tyr Lys Tyr Val Tyr Glu Thr Val Val Val
35 40 45
Asp Ser Asp Gly His Thr Val Thr Pro Ala Ala Ser Glu Val Ala Thr
50 55 60
Ala Ala Thr Ser Ala Ile Ile Thr Thr Ser Val Leu Ala Pro Thr Ser
65 70 75 80
Ser Ala Ala Ala Gly Ile Ala Ala Ser Ile Ala Val Ser Ser Ala Ala
85 90 95
Leu Ala Lys Asn Glu Lys Ile Ser Asp Ala Ala Ala Ser Ala Thr Ala
100 105 110
Ser Thr Ser Gln Gly Ala Ser Ser Ser Ser Leu Ala Ala Ser Ala
115 120 125
<210>32
<211>618
<212>DNA
<213>Artificial sequence
<220>
<223>TFP13-aa
<220>
<221>misc_feature
<222>(42)..(42)
<223>n is a,c,g,or t
<400>32
ggccattacg gccggggatt caaatatata tatctactca gnttgaataa gacactatag 60
caagaccatt tgaactgaaa gaaacagttt ctttgctccc ctctcgaatt ccaactattt 120
acagtccttc ctttataaaa attaactagc gagcaagaaa acatttgttt agtgctaccc 180
aactacttac attcctttaa aaaccacaat atttaagtta acctgagctt tatttttaaa 240
atgaaattct caactgccgt tactacgttg attagttctg gtgccatcgt gtctgcttta 300
ccacacgtgg atgttcacca agaagatgcc caccaacata agagggccgt tgcgtacaaa 360
tacgtttacg aaactgttgt tgtcgattct gatggccaca ctgtaactcc tgctgcttca 420
gaagtcgcta ctgctgctac ctctgctatc attacaacat ctgtgttggc tccaacctcc 480
tccgcagccg ctgggatagc cgcttccatt gctgtttcat ctgctgcctt agccaagaat 540
gagaaaatct ctgatgccgc tgcatctgcc actgcctcaa catctcaagg ggcatcctcc 600
tcctccctgg ccgcctcg 618
<210>33
<211>68
<212>PRT
<213>Artificial sequence
<220>
<223>TFP17-AA
<400>33
Met Lys Phe Ser Ser Val Thr Ala Ile Thr Leu Ala Thr Val Ala Thr
1 5 10 15
Val Ala Thr Ala Lys Lys Gly Glu His Asp Phe Thr Thr Thr Leu Thr
20 25 30
Leu Ser Ser Asp Gly Ser Leu Thr Thr Thr Thr Ser Thr His Thr Thr
35 40 45
His Lys Tyr Gly Lys Phe Asn Lys Thr Ser Lys Ser Lys Thr Pro Trp
50 55 60
Ala Ala Ser Ala
65
<210>34
<211>391
<212>DNA
<213>Artificial sequence
<220>
<223>TFP17-nt
<400>34
ggccattacg gccggggttt ctttctcttt tttctttttt gaataaagaa ttttccttta 60
aggagtaact taagcattta gctgcacatt aaacactttt ttttttactt ctaactcaca 120
cacttttgga agaacattta ttttttcgac cttctttccc aaatacccag cgctttataa 180
ttgaaatatg aagttctctt ctgttactgc tattactcta gccaccgttg ccaccgttgc 240
cactgctaag aagggtgaac atgatttcac taccacttta actttgtcat cggacggtag 300
tttaactact accacctcta ctcataccac tcacaagtat ggtaagttca acaagacttc 360
caagtccaag accccctggg ccgcctcggc c 391
<210>35
<211>199
<212>PRT
<213>Artificial sequence
<220>
<223>TFP18-aa
<400>35
Met Gln Phe Lys Asn Ala Leu Thr Ala Thr Ala Ile Leu Ser Ala Ser
1 5 10 15
Ala Leu Ala Asn Ser Thr Thr Ser Ile Pro Ser Ser Cys Ser Ile Gly
20 25 30
Thr Ser Ala Thr Ala Thr Ala Gln Ala Asp Leu Asp Lys Ile Ser Gly
35 40 45
Cys Ser Thr Ile Val Gly Asn Leu Thr Ile Thr Gly Asp Leu Gly Ser
50 55 60
Ala Ala Leu Ala Ser Ile Gln Glu Ile Asp Gly Ser Leu Thr Ile Phe
65 70 75 80
Asn Ser Ser Ser Leu Ser Ser Phe Ser Ala Asp Ser Ile Lys Lys Ile
85 90 95
Thr Gly Asp Leu Asn Met Gln Glu Leu Ile Ile Leu Thr Ser Ala Ser
100 105 110
Phe Gly Ser Leu Gln Glu Val Asp Ser Ile Asn Met Val Thr Leu pro
115 120 125
Ala Ile Ser Thr Phe Ser Thr Asp Leu Gln Asn Ala Asn Asn Ile Ile
130 135 140
Val Ser Asp Thr Thr Leu Glu Ser Val Glu Gly Phe Ser Thr Leu Lys
145 150 155 160
Lys Val Asn Val Phe Asn Ile Asn Asn Asn Arg Tyr Leu Asn Ser Phe
165 170 175
Gln Ser Ser Leu Glu Ser Val Ser Asp Ser Leu Gln Phe Ser Ser Asn
180 185 190
Gly Asp Leu Ala Ala Ser Ala
195
<210>36
<211>760
<212>DNA
<213>Artificial sequence
<220>
<223>TFP18-nt
<400>36
ggccattacg gccgaaaaga aattaaataa aagagttaat cgttcattcg cttctacaca 60
gtttaatctt ttccattttt ctttcaacaa gtccctttga gctatcaaga atacgtttat 120
ttgactttta aagatctagt tttaatttta ctattattcc gcaatgcaat tcaagaacgc 180
tttgactgct actgctattc taagtgcctc cgctctagct aactcaacta cttctattcc 240
atcttcatgt agtattggta cttctgccac tgctactgct caagctgatt tggacaaaat 300
ctccggttgt agtaccattg ttggtaactt gaccatcacc ggtgacttgg gttccgctgc 360
tttggctagt atccaagaga ttgatggttc cttgactatc ttcaactcca gttctttatc 420
ttctttctcc gctgactcta tcaagaaaat caccggtgat ttgaacatgc aagaattgat 480
cattttgacc agtgcttctt tcggttcttt gcaagaagta gactccatta acatggtgac 540
tttgcctgcc atttctacct tctccaccga tttacaaaat gctaacaaca ttattgtttc 600
tgacaccact ttggaaagtg tcgaaggttt ctccactttg aagaaggtta atgtttttaa 660
catcaacaac aacagatatc taaactcttt ccaatcttcc ttggaaagtg tctctgactc 720
tttacaattc tcttccaacg gtgacctggc cgcctcggcc 760
<210>37
<211>148
<212>PRT
<213>Artificial sequence
<220>
<223>TFP19-aa
<400>37
Met Val Ser Lys Thr Trp Ile Cys Gly Phe Ile Ser Ile Ile Thr Val
1 5 10 15
Val Gln Ala Leu Ser Cys Glu Lys His Asp Val Leu Lys Lys Tyr Gln
20 25 30
Val Gly Lys Phe Ser Ser Leu Thr Ser Thr Glu Arg Asp Thr Pro Pro
35 40 45
Ser Thr Thr Ile Glu Lys Trp Trp Ile Asn Val Cys Glu Glu His Asn
50 55 60
Val Glu Pro Pro Glu Glu Cys Lys Lys Asn Asp Met Leu Cys Gly Leu
65 70 75 80
Thr Asp Val Ile Leu Pro Gly Lys Asp Ala Ile Thr Thr Gln Ile Ile
85 90 95
Asp Phe Asp Lys Asn Ile Gly Phe Asn Val Glu Glu Thr Glu Ser Ala
100 105 110
Leu Thr Leu Thr Leu Lys Gly Ala Thr Trp Gly Ala Asn Ser Phe Asp
115 120 125
Ala Lys Leu Glu phe Gln Cys Asn Asp Asn Met Lys Gln Asp Glu Leu
130 135 140
Ala Ala Ser Ala
145
<210>38
<211>464
<212>DNA
<213>Artificial sequence
<220>
<223>TFP19-nt
<400>38
ggccattacg gccggggacg atggtatcga agacttggat atgtggcttc atcagtataa 60
ttacagtggt acaggccttg tcctgcgaga agcatgatgt attgaaaaag tatcaggtgg 120
gaaaatttag ctcactaact tctacggaaa gggatactcc gccaagcaca actattgaaa 180
agtggtggat aaacgtttgc gaagagcata acgtagaacc tcctgaagaa tgtaaaaaaa 240
atgacatgct atgtggttta acagatgtca tcttgcccgg taaggatgct atcaccactc 300
aaattataga ttttgacaaa aacattggct tcaatgtcga ggaaactgag agtgcgctta 360
cattgacact aaaaggcgct acgtggggcg ccaattcttt tgacgcaaaa ctagaatttc 420
agtgtaatga caatatgaaa caagacgaac tggccgcctc ggcc 464
<210>39
<211>187
<212>PRT
<213>Artificial sequence
<220>
<223>TFP20-aa
<400>39
Met Leu Phe Lys Ser Leu Ser Lys Leu Ala Thr Ala Ala Ala Phe Phe
1 5 10 15
Ala Gly Val Ala Thr Ala Asp Asp Val Pro Ala Ile Glu Val Val Gly
20 25 30
Asn Lys Phe Phe Tyr Ser Asn Asn Gly Ser Gln Phe Tyr Ile Arg Gly
35 40 45
Val Ala Tyr Gln Ala Asp Thr Ala Asn Glu Thr Ser Gly Ser Thr Val
50 55 60
Asn Asp Pro Leu Ala Asn Tyr Glu Ser Cys Ser Arg Asp Ile Pro Tyr
65 70 75 80
Leu Lys Lys Leu Asn Thr Asn Val Ile Arg Val Tyr Ala Ile Asn Thr
85 90 95
Thr Leu Asp His Ser Glu Cys Met Lys Ala Leu Asn Asp Ala Asp Ile
100 105 110
Tyr Val Ile Ala Asp Leu Ala Ala Pro Ala Thr Ser Ile Asn Arg Asp
115 120 125
Asp Pro Thr Trp Thr Val Asp Leu Phe Asn Ser Tyr Lys Thr Val Val
130 135 140
Asp Thr Phe Ala Asn Tyr Thr Asn Val Leu Gly Phe Phe Ala Gly Asn
145 150 155 160
Glu Val Thr Asn Asn Tyr Thr Asn Thr Asp Ala Ser Ala Phe Val Lys
165 170 175
Ala Ala Ile Arg Asp Val Leu Ala Ala Ser Ala
180 185
<210>40
<211>664
<212>DNA
<213>Artificial sequence
<220>
<223>TFP20-nt
<400>40
ggccattacg gccggggtgt cgttttatta agctatttca aaatcagttt ttatttttaa 60
agtctgataa aacaaaaaca acaaacacag ctaaatctca acaatgttgt ttaaatccct 120
ttcaaagtta gcaaccgctg ctgctttttt tgctggcgtc gcaactgcgg acgatgttcc 180
agcgattgaa gttgttggta ataagttttt ctactccaac aacggtagtc agttctacat 240
aagaggtgtt gcttatcagg ctgataccgc taatgaaact agcggatcta ctgtcaacga 300
tcctttggcc aattatgaga gttgttccag agatattcca tacctcaaaa aattgaacac 360
aaatgttatc cgtgtctacg ctatcaatac cactctagat cactccgaat gtatgaaggc 420
tttgaatgat gctgacatct atgtcatcgc tgatttagca gctccagcca cctctatcaa 480
tagagacgat ccaacttgga ctgttgactt gttcaacagc tacaaaaccg ttgttgacac 540
ttttgctaat tacaccaacg ttttgggttt cttcgccggt aatgaagtta ctaacaatta 600
caccaacaca gatgcatctg ctttcgtgaa ggcagctatt agagacgtcc tggccgcctc 660
ggcc 664
<210>41
<211>55
<212>PRT
<213>Artificial sequence
<220>
<223>TFP21-aa
<400>41
Met Leu Gln Ser Val Val Phe Phe Ala Leu Leu Thr Phe Ala Ser Ser
1 5 10 15
Val Ser Ala Ile Tyr Ser Asn Asn Thr Val Ser Thr Thr Thr Thr Leu
20 25 30
Ala Pro Ser Tyr Ser Leu Val Pro Gln Glu Thr Thr Ile Ser Tyr Ala
35 40 45
Asp Asp Leu Ala Ala Ser Ala
50 55
<210>42
<211>407
<212>DNA
<213>Artificial sequence
<220>
<223>TFP21-nt
<400>42
ggccattacg gccggggaag caactagttt agcacaacat ccaaccaaga ggtttctcgc 60
gtatttctct cattttttta cccattttac aaattttttt tgctatttga gccatagtac 120
ccattaatag gtctcgtcca ttcccttgtt ttttttttat tgtttcaatt acactacata 180
attaaaaatc acatcacttt cactctcacc ttagtcgttc tttatcaacc aaaaataaaa 240
aaatgcttca atccgttgtc tttttcgctc ttttaacctt cgcaagttct gtgtcagcga 300
tttattcaaa caatactgtt tctacaacta ccactttagc gcccagctac tccttggtgc 360
cccaagagac taccatatcg tacgccgacg acctggccgc ctcggcc 407
<210>43
<211>190
<212>PRT
<213>Artificial sequence
<220>
<223>TFP25-aa
<400>43
Met Asn Trp Leu Phe Leu Val Ser Leu Val Phe Phe Cys Gly Val Ser
1 5 10 15
Thr His Pro Ala Leu Ala Met Ser Ser Asn Arg Leu Leu Lys Leu Ala
20 25 30
Asn Lys Ser Pro Lys Lys Ile Ile Pro Leu Lys Asp Ser Ser Phe Glu
35 40 45
Asn Ile Leu Ala Pro Pro His Glu Asn Ala Tyr Ile Val Ala Leu Phe
50 55 60
Thr Ala Thr Ala pro Glu Ile Gly Cys Ser Leu Cys Leu Glu Leu Glu
65 70 75 80
Ser Glu Tyr Asp Thr Ile Val Ala Ser Trp Phe Asp Asp His Pro Asp
85 90 95
Ala Lys Ser Ser Asn Ser Asp Thr Ser Ile Phe Phe Thr Lys Val Asn
100 105 110
Leu Glu Asp Pro Ser Lys Thr Ile ProLys Ala Phe Gln Phe Phe Gln
115 120 125
Leu Asn Asn Val Pro Arg Leu Phe Ile Phe Lys Pro Asn Ser Pro Ser
130 135 140
Ile Leu Asp His Ser Val Ile Ser Ile Ser Thr Asp Thr Gly Ser Glu
145 150 155 160
Arg Met Lys Gln Ile Ile Gln Ala Ile Lys Gln Phe Ser Gln Val Asn
165 170 175
Asp Phe Ser Leu His Leu Pro Val Gly Leu Ala Ala Ser Ala
180 185 190
<210>44
<211>654
<212>DNA
<213>Artificial sequence
<220>
<223>TFP25-nt
<400>44
ggccattacg gccgggggaa taccaggcac acgctcttcg aacactgaac cacacgcgtc 60
cgcatcaaac tcttcctccc aaacatgaat tggctgtttt tggtctcgct ggttttcttc 120
tgcggcgtgt caacccatcc tgccctggca atgtccagca acagactact aaagctggct 180
aataaatctc ccaagaaaat tatacctctg aaggactcaa gttttgaaaa catcttggca 240
ccacctcacg aaaatgccta tatagttgct ctgtttactg ccacagcgcc cgaaattggc 300
tgttctctgt gtctcgagct agaatccgaa tacgacacca tagtggcctc ctggtttgat 360
gatcatccgg atgcaaaatc gtccaattcc gatacatcta ttttcttcac aaaggtcaat 420
ttggaggacc cttctaagac cattcctaaa gcgttccagt ttttccaact aaacaatgtt 480
cctagattgt tcatcttcaa accaaactct ccctctattc tggaccacag cgtgatcagt 540
atttccactg atactggctc agaaagaatg aagcaaatca tacaagccat taagcagttc 600
tcgcaagtaa acgacttctc tttacactta cctgtgggtc tggccgcctc ggcc 654
<210>45
<211>89
<212>PRT
<213>Artificial sequence
<220>
<223>TFP27-aa
<400>45
Met Lys Leu Ser Ala Leu Leu Ala Leu Ser Ala Ser Thr Ala Val Leu
1 5 10 15
Ala Ala Pro Ala Val His His Ser Asp Asn His His His Asn Asp Lys
20 25 30
Arg Ala Val Val Thr Val Thr Gln Tyr Val Asn Ala Asp Gly Ala Val
35 40 45
Val Ile Pro Ala Ala Thr Thr Ala Thr Ser Ala Ala Ala Asp Gly Lys
50 55 60
Val Glu Ser Val Ala Ala Ala Thr Thr Thr Leu Ser Ser Thr Ala Ala
65 70 75 80
Ala Ala Thr Thr Leu Ala Ala Ser Ala
85
<210>46
<211>470
<212>DNA
<213>Artificial sequence
<220>
<223>TFP27-nt
<400>46
ggccattacg gccggggacg ctcctttcat cggtaactaa gaagaaaaaa aaaaaagtac 60
gaccacacaa tttccagtgt attcattcct taaacttcgt ttatttttta ttcattcatt 120
catttttatt tgaatataac caactactag tccttccttt aaacaaaaat ttaccctccc 180
ttaatttttc aagaaattcc agtatgaaat tatccgctct attagcttta tcagcctcca 240
ccgccgtctt ggccgctcca gctgtccacc atagtgacaa ccaccaccac aacgacaagc 300
gtgccgttgt caccgttact cagtacgtca acgcagacgg cgctgttgtt attccagctg 360
ccaccaccgc tacctcggcg gctgctgatg gaaaggtcga gtctgttgct gctgccacca 420
ctactttgtc ctcgactgcc gccgccgcta caaccctggc cgcctcggcc 470
<210>47
<211>33
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-YGR-F
<400>47
ccggatccat ggtgttcggt cagctgtatg ccc 33
<210>48
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-SIM-F
<400>48
cggatccatg aaattctcaa ctgccgttac tacg 34
<210>49
<211>31
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-YNL-F
<400>49
ccggatccat gaagttctct tctgttactg c 31
<210>50
<211>31
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-ECM-F
<400>50
ccggatccat gcaattcaag aacgctttga c 31
<210>51
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-ATG-F
<400>51
ccggatccat ggtatcgaag acttggatat gtgg 34
<210>52
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-GAS-F
<400>52
ccggatccat gttgtttaaa tccctttcaa agttagc 37
<210>53
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-YOR-F
<400>53
ccggatccat gcttcaatcc gttgtctttt tcgc 34
<210>54
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-OST-F
<400>54
ccggatccat gaattggctg tttttggtct cgctgg 36
<210>55
<211>32
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-UTH-F
<400>55
ccggatccat gtgtttcctt ctcgagacct cg 32
<210>56
<211>43
<212>DNA
<213>Artificial sequence
<220>
<223>IL2-TGA-R
<400>56
gtcactccgt tcaagtcgac tcaagttagt gttgagatga tgc 43
<210>57
<211>24
<212>DNA
<213>Artificial sequence
<220>
<223>Sac-GAL-F
<400>57
gagctcatcg cttcgctgat taat 24
<210>58
<211>27
<212>DNA
<213>Artificial sequence
<220>
<223>GAL-BamH-R
<400>58
ggatcctgaa ttttcaaaaa ttcttac 27
<210>59
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>KR-IL32Alpha-F
<400>59
ctcgccttag ataaaagaat gtgcttcccg aaggtcct 38
<210>60
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>IL32-INV-R
<400>60
ctcgccttag ataaaagaat gtgcttcccg aaggtcct 38
<210>61
<211>187
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-11-AA
<400>61
Met Lys Leu Ser Thr Val Leu Leu Ser Ala Gly Leu Ala Ser Thr Thr
1 5 10 15
Leu Ala Gln Phe Ser Asn Ser Thr Ser Ala Ser Ser Thr Asp Val Thr
20 25 30
Ser Ser Ser Ser Ile Ser Thr Ser Ser Gly Ser Val Thr Ile Thr Ser
35 40 45
Ser Glu Ala Pro Glu Ser Asp Asn Gly Thr Ser Thr Ala Ala Pro Thr
50 55 60
Glu Thr Ser Thr Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr
65 70 75 80
Ser Thr Glu Ala Pro Thr Thr Ala Ile Pro Thr Asn Gly Thr Ser Thr
85 90 95
Glu Ala Pro Thr Asp Thr Thr Thr Glu Ala Pro Thr Thr Ala Leu Pro
100 105 110
Thr Asn Gly Thr Ser Thr Glu Ala Pro Thr Asp Thr Thr Thr Glu Ala
115 120 125
Pro Thr Thr Gly Leu Pro Thr Asn Gly Thr Thr Ser Ala Phe Pro Pro
130 135 140
Thr Thr Ser Leu Pro Pro Ser Asn Thr Thr Thr Thr Pro Pro Tyr Asn
145 150 155 160
Pro Ser Thr Asp Tyr Thr Thr Asp Tyr Thr Val Val Thr Glu Tyr Thr
165 170 175
Thr Tyr Cys Pro Glu Pro Leu Ala Ala Ser Ala
180 185
<210>62
<211>621
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-11-NT
<400>62
ggccattacg gccggggagc tacaaagaca agcaaaataa aatacgttcg ctctattaag 60
atgaaattat caactgtcct attatctgcc ggtttagcct cgactacttt ggcccaattt 120
tccaacagta catctgcttc ttccaccgat gtcacttcct cctcttccat ctccacttcc 180
tctggctcag taactatcac atcttctgaa gctccagaat ccgacaacgg taccagcaca 240
gctgcaccaa ctgaaacctc aacagaggct ccaaccactg ctatcccaac taacggtacc 300
tctactgaag ctccaaccac tgctatccca actaacqgta cctctactga agctccaact 360
gatactacta ctgaagctcc aaccaccgct cttccaacta acggtacttc tactgaagct 420
ccaactgata ctactactga agctccaacc accggtcttc caaccaacgg taccacttca 480
gctttcccac caactacatc tttgccacca agcaacacta ccaccactcc tccttacaac 540
ccatctactg actacaccac tgactacact gtagtcactg aatatactac ttactgtccg 600
gaaccactgg ccgcctcggc c 621
<210>63
<211>165
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-22-AA
<400>63
Met Gln Tyr Lys Lys Thr Leu Val Ala Ser Ala Leu Ala Ala Thr Thr
1 5 10 15
Leu Ala Ala Tyr Ala Pro Ser Glu Pro Trp Ser Thr Leu Thr Pro Thr
20 25 30
Ala Thr Tyr Ser Gly Gly Val Thr Asp Tyr Ala Ser Thr Phe Gly Ile
35 40 45
Ala Val Gln Pro Ile Ser Thr Thr Ser Ser Ala Ser Ser Ala Ala Thr
50 55 60
Thr Ala Ser Ser Lys Ala Lys Arg Ala Ala Ser Gln Ile Gly Asp Gly
65 70 75 80
Gln Val Gln Ala Ala Thr Thr Thr Ala Ser Val Ser Thr Lys Ser Thr
85 90 95
Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln Ala Thr Thr
100 105 110
Lys Thr Thr Ala Ala Ala Val Ser Gln Ile Gly Asp Gly Gln Ile Gln
115 120 125
Ala Thr Thr Lys Thr Thr Ser Ala Lys Thr Thr Ala Ala Ala Val Ser
130 135 140
Gln Ile Ser Asp Gly Gln Ile Gln Ala Thr Thr Thr Thr Leu Ala Pro
145 150 155 160
Leu Ala Ala Ser Ala
165
<210>64
<211>564
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-22-NT
<400>64
ggccattacg gccgggggaa taagaaactc atattccttt tctaacccta gtacaataat 60
aataatataa tgcaatacaa aaagactttg gttgcctctg ctttggccgc tactacattg 120
gccgcctatg ctccatctga gccttggtcc actttgactc caacagccac ttacagcggt 180
ggtgttaccg actacgcttc caccttcggt attgccgttc aaccaatctc cactacatcc 240
agcgcatcat ctgcagccac cacagcctca tctaaggcca agagagctgc ttcccaaatt 300
ggtgatggtc aagtccaagc tgctaccact actgcttctg tctctaccaa gagtaccgct 360
gccgccgttt ctcagatcgg tgatggtcaa atccaagcta ctaccaagac taccgctgct 420
gctgtctctc aaattggtga tggtcaaatt caagctacca ccaagactac ctctgctaag 480
actaccgccg ctgccgtttc tcaaatcagt gatggtcaaa tccaagctac caccactact 540
ttagcccctc tggccgcctc ggcc 564
<210>65
<211>48
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-29-AA
<400>65
Met Lys Leu Glu Asn Thr Leu Phe Thr Leu Gly Ala Leu Gly Ser Ile
1 5 10 15
Ser Ala Ala Leu Val Ile Pro Asn Leu Glu Asn Ala Ala Asp His His
20 25 30
Glu Leu Ile Asn Lys Glu Asp His His Glu Arg Leu Ala Ala Ser Ala
35 40 45
<210>66
<211>216
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-29-NT
<400>66
ggccattacg gccggggaat tagcttcatc gccaataaaa aaacaaacta aacctaattc 60
taacaagcaa agatgaagtt agaaaatact ctatttacac tcggtgccct agggagcatc 120
tctgctgctt tggtcatccc aaatcttgaa aatgccgccg accaccacga actgattaac 180
aaggaagatc accacgagag actggccgcc tcggcc 216
<210>67
<211>208
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-34-AA
<400>67
Met Arg Ala Thr Thr Leu Leu Ser Ser Val Val Ser Leu Ala Leu Leu
1 5 10 15
Ser Lys Glu Val Leu Ala Thr Pro Pro Ala Cys Leu Leu Ala Cys Val
20 25 30
Ala Gln Val Gly Lys Ser Ser Ser Thr Cys Asp Ser Leu Asn Gln Val
35 40 45
Thr Cys Tyr Cys Glu His Glu Asn Ser Ala Val Lys Lys Cys Leu Asp
50 55 60
Ser Ile Cys Pro Asn Asn Asp Ala Asp Ala Ala Tyr Ser Ala Phe Lys
65 70 75 80
Ser Ser Cys Ser Glu Gln Asn Ala Ser Leu Gly Asp Ser Ser Ser Ser
85 90 95
Ala Ser Ser Ser Ala Ser Ser Ser Ser Lys Ala Ser Ser Ser Thr Lys
100 105 110
Ala Ser Ser Ser Ser Ala Ser Ser Ser Thr Lys Ala Ser Ser Ser Ser
115 120 125
Ala Ser Ser Pro Thr Lys Ala Ser Ser Ser Ser Ala Ala Pro Ser Ser
130 135 140
Ser Lys Ala Ser Ser Thr Glu Ser Ser Ser Ser Ser Ser Ser Ser Thr
145 150 155 160
Lys Ala Pro Ser Ser Glu Glu Ser Ser Ser Thr Tyr Val Ser Ser Ser
165 170 175
Lys Gln Ala Ser Ser Thr Ser Glu Ala His Ser Ser Ser Ala Ala Ser
180 185 190
Ser Thr Val Ser Gln Glu Thr Val Ser Ser Ala Leu Ala Ala Ser Ala
195 200 205
<210>68
<211>694
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-34-NT
<400>68
ggccattacg gccgggaggg tcaaagctca cagcactact acactcgttc aacactcgtt 60
atatattatc atgcgcgcca ccactttatt atcttcagtc gtttctttgg cattgttgtc 120
gaaggaagtc ttagcaacac ctccagcttg tttattggcc tgtgttgcgc aagtcggcaa 180
atcctcttcc acatgtgact ctttgaatca agtcacctgt tactgtgaac acgaaaactc 240
cgccgtcaag aaatgtctag actccatctg cccaaacaat gacgctgatg ctgcttattc 300
tgctttcaag agttcttgtt ccgaacaaaa tgcttcattg ggcgattcca gcagcagtgc 360
ctcctcatcc gcttcttcat ccagcaaggc ctcttcttct accaaggctt cttccagtag 420
cgcttcctcc tctaccaagg cttcttccag tagcgcttcc tcccctacta aagcttcttc 480
cagcagcgct gccccatctt ctagcaaggc ttcttccacc gaatcctctt cttcctcttc 540
ttcttccacc aaggctcctt ccagtgaaga atcctcttcc acttatgtct cttcgagcaa 600
gcaagcttcc tccactagcg aggctcactc ttccagtgct gcctcttcga ccgtgtccca 660
agaaacagtc tcctctgctc tggccgcctc ggcc 694
<210>69
<211>38
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-38-AA
<400>69
Met Lys Leu Ser Gln Val Val Val Ser Ala Val Ala Phe Thr Gly Leu
1 5 10 15
Val Ser Ala Ala Asn Ser Ser Asn Ser Ser Ser Ser Lys Asn Ala Ala
20 25 30
Gln Leu Ala Ala Ser Ala
35
<210>70
<211>184
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-38-NT
<400>70
ggccattacg gccgggggac tatcaaatca tacagatatt gtcaaaaaaa aaaaagacta 60
ataataaaaa atgaagttat ctcaagttgt tgtttccgcc gtcgccttca ctggtttagt 120
aagtgctgct aacagttcta acagctcaag ctcaaagaat gctgcccaac tggccgcctc 180
ggcc 184
<210>71
<211>26
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-CIS-F
<400>71
ccggatccat gcaattcaaa aacgtc 26
<210>72
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-SED-F
<400>72
ccggatccat gaaattatca actgtcctat tatctgc 37
<210>73
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-SIM-F
<400>73
ccggatccat gaaattctca actgccgtta ctacg 35
<210>74
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-YOR247W-F
<400>74
ccggatccat gcttcaatcc gttgtctttt tcgc 34
<210>75
<211>30
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-HSP-F
<400>75
ccggatccat gcaatacaaa aagactttgg 30
<210>76
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>BamH-OST-F
<400>76
ccggatccat gaattggctg tttttggtct cgctgg 36
<210>77
<211>41
<212>DNA
<213>Artificial sequence
<220>
<223>IL32-TGA-R
<400>77
cactccgttc aagtcgactc attttgagga ttggggttca g 41
<210>78
<211>30
<212>DNA
<213>Artificial sequence
<220>
<223>H77-1-R
<400>78
aagtcgacat ttaaatcttt tatctaaggc 30
<210>79
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>hGH-F
<400>79
ttcccaacca ttcccttatc 20
<210>80
<211>19
<212>DNA
<213>Artificial sequence
<220>
<223>hGH-R
<400>80
ctagaagcca cagctgccc 19
<210>81
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>KR-hGH-F
<400>81
ctcgccttag ataaaagatt cccaaccatt cccttatc 38
<210>82
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>hGH-Sal-R
<400>82
cactccgttc aagtcgacct agaagccaca gctgccc 37
<210>83
<211>70
<212>DNA
<213>Artificial sequence
<220>
<223>GT70-R
<400>83
tcagatttac agataatgat gtcattatta aatatatata tatatatatt gtcactccgt 60
tcaagtcgac 70
<210>84
<211>101
<212>PRT
<213>Artificial sequence
<220>
<223>PpTFP1-aa
<400>84
Met Gln Phe Asn Ser Val Val Ile Ser Gln Leu Leu Leu Thr Leu Ala
1 5 10 15
Ser Val Ser Met Gly Ala Ser Thr Ala Phe Lys Glu His His Gln His
20 25 30
Gln Arg Ala Thr Leu Glu Lys Arg Ala Thr Thr Cys Lys Phe Pro Thr
35 40 45
Asp Lys Asn Leu Val Ala Val Thr Pro Asn Ser Lys Asn Gly Gly Trp
50 55 60
Ala Leu Ser Pro Asp Gln Glu Cys Thr Ala Gly Ser Tyr Cys Pro Tyr
65 70 75 80
Ala Cys Pro Pro Gly Gln Leu Met Ala Gln Trp Asp Pro Ser Ala Thr
85 90 95
Leu Ala Ala Ser Ala
100
<210>85
<211>419
<212>DNA
<213>Artificial sequence
<220>
<223>PpTFP1-nt
<400>85
ggccattacg ccggggcaca gtaactttga cataatatct ggtagctgca tcacttcacc 60
gactattcat tccttccttt ttagtattac caactatatc acattccttt aagaaaatgc 120
aattcaacag tgtcgtcatc agccaacttt tgctgactct agccagtgtc tcaatgggag 180
cttcaaccgc tttcaaggag caccaccagc accaaagagc tactctagag aagagagcta 240
ctacctgcaa attccccact gacaaaaact tggtcgctgt tacaccaaac tccaaaaatg 300
gaggctgggc tctgagtcca gaccaggagt gcacagcagg ttcttattgt ccttatgctt 360
gtccaccagg ccagttgatg gctcaatggg acccatcggc cacactggcc gcctcggcc 419
<210>86
<211>94
<212>PRT
<213>Artificial sequence
<220>
<223>PpTFP2-aa
<400>86
Met Gln Phe Ser Ile Val Ala Thr Leu Ala Leu Ala Gly Ser Ala Leu
1 5 10 15
Ala Ala Tyr Ser Asn Val Thr Tyr Thr Tyr Glu Thr Thr Ile Thr Asp
20 25 30
Val Val Thr Glu Leu Thr Thr Tyr Cys Pro Glu Pro Thr Thr Phe Val
35 40 45
His Lys Asn Lys Thr Ile Thr Val Thr Ala Pro Thr Thr Leu Thr Ile
50 55 60
Thr Asp Cys Pro Cys Thr Ile Ser Lys Thr Thr Lys Ile Thr Thr Asp
65 70 75 80
Val Pro Pro Thr Thr His Ser Thr Pro Leu Ala Ala Ser Ala
85 90
<210>87
<211>345
<212>DNA
<213>Artificial sequence
<220>
<223>PpTFP2-nt
<400>87
ggccattacg gccgggggac ttacatttta ccgttccgtc actcgcttca ctcaacaaca 60
aaaatgcaat tctctatcgt cgctactttg gctcttgctg gttccgctct ggctgcttac 120
tctaacgtaa cttacactta cgagactacc atcaccgatg ttgtcaccga gctcaccact 180
tactgcccag agccaaccac cttcgttcac aagaacaaga ccatcactgt gaccgcccca 240
accactttga ccatcactga ctgtccttgc accatctcca agaccaccaa gatcaccact 300
gatgttccac caaccaccca ctccacccca ctggccgcct cggcc 345
<210>88
<211>82
<212>PRT
<213>Artificial sequence
<220>
<223>PpTFP3-aa
<400>88
Met Lys Phe Ser Thr Ala Phe Ala Gly Phe Val Ala Leu Asn Ala Val
1 5 10 15
Ser Ile Val Ala Gln Asp Glu Ala Thr Asp Ala His Val Val Thr Thr
20 25 30
Thr Val Thr Thr Ala Ser Thr Glu Thr His Arg Trp Gly Arg Phe Asp
35 40 45
Lys Thr Ser Pro Pro Thr Thr Ser Thr Ser Ser Gly Thr His Arg Trp
50 55 60
Gly Arg Phe Asn Lys Thr Pro Asp Pro Thr Thr Thr Thr Ser Ala Ala
65 70 75 80
Ser Ala
<210>89
<211>273
<212>DNA
<213>Artificial sequence
<220>
<223>PpTFp3-nt
<400>89
ggccattacg gccggggaac atcaagaatg aagttttcca ctgcgtttgc tggctttgtt 60
gccctaaatg ctgtgtccat tgttgctcag gacgaggcta ccgatgctca cgttgtcacc 120
acaactgtga ccaccgcttc cactgagact cacagatggg gaagattcga caagacttct 180
cctcctacaa cttccacttc ttcaggtact cacagatggg gaagatttaa caaaactcca 240
gatcctacca ctaccacctc ggccgcctcg gcc 273
<210>90
<211>127
<212>PRT
<213>Artificial sequernce
<220>
<223>PpTFP4-aa
<400>90
Met Gln Tyr Arg Ser Leu phe Leu Gly Ser Ala Leu Leu Ala Ala Ala
1 5 10 15
Asn Ala Ala Val Tyr Asn Thr Thr Val Thr Asp Val Val Ser Glu Leu
20 25 30
Glu Thr Thr Val Leu Thr Ile Thr Ser Cys Ala Glu Asp Lys Cys Ile
35 40 45
Thr Ser Lys Ser Thr Gly Leu Ile Thr Thr Ser Thr Leu Thr Lys His
50 55 60
Gly Val Val Thr Val Val Thr Thr Val Cys Asp Leu Pro Ser Thr Thr
65 70 75 80
Lys Ser Tyr Val Pro Pro Ala Lys Thr Thr Thr Ile Pro Pro Pro Glu
85 90 95
Lys Thr Thr Thr Thr Val Pro Pro Pro Ala Lys Thr Thr Thr Thr Val
100 105 110
Pro Pro Pro Ala Lys Thr Thr Ser Thr Ala Leu Ala Ala Ser Ala
115 120 125
<210>91
<211>444
<212>DNA
<213>Artificial sequence
<220>
<223>PpTFP4-nt
<400>91
ggccattacg gggggaactc actgtttcag tttattccaa ctactttcac tcacttatca 60
aaaatgcaat acagatctct ctttttaggt tccgccttat tggccgctgc taacgctgct 120
gtttacaaca ccaccgtcac tgacgttgtt tccgagttgg agaccaccgt tctgactatc 180
acctcttgtg ctgaggacaa gtgtatcacc agtaagtcca ccggattgat cactacctcc 240
accctcacca agcacggtgt tgtcactgtt gtcaccactg tctgtgactt gccaagcacc 300
accaagagct acgtcccacc tgctaagact actactattc ctcctccaga gaagactacc 360
accactgtcc cacctccagc caagactacc accactgtcc cacctccagc caagactact 420
agtaccgccc tggccgcctc ggcc 444
<210>92
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>YGR279C-F
<400>92
ggccattacg gccaaaatgc gtctctctaa cctaattg 38
<210>93
<211>21
<212>DNA
<213>Artificial sequence
<220>
<223>YGR279C-R
<400>93
tcattggata gaatacccca g 21
<210>94
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>YLR037C-F
<400>94
ggccattacg gccaaaatgg tcaaactaac ttcaattg 38
<210>95
<211>22
<212>DNA
<213>Artificial sequence
<220>
<223>YLR037C-R
<400>95
ttagtttgga acagcagtgt ag 22
<210>96
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>YLR110C-F
<400>96
ggccattacg gccaaaatgc aattttctac tgtcgc 36
<210>97
<211>20
<212>DNA
<213>Arti ficial sequence
<220>
<223>YLR110C-R
<400>97
ttacaacaac aaagcagcgg 20
<210>98
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>YOR383C-F
<400>98
ggccattacg gccaaaatga aattctcttc cgctttg 37
<210>99
<211>21
<212>DNA
<213>Artificial sequence
<220>
<223>YOR383C-R
<400>99
ttacaataac atgacggcag c 21
<210>100
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>YIL011W-F
<400>100
ggccattacg gccaaaatgt ctttcactaa aatcgc 36
<210>101
<211>21
<212>DNA
<213>Artificial sequence
<220>
<223>YIL011W-R
<400>101
tcataagagc atagcagcgg c 21
<210>102
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>YHR214W-F
<400>102
ggccattacg gccaaaatgt tcaatcgttt taacaaat 38
<210>103
<211>22
<212>DNA
<213>Artificial sequence
<220>
<223>YHR214W-R
<400>103
ttacaaaccg gaaacagaac ca 22
<210>104
<211>39
<212>DNA
<213>Artificial sequence
<220>
<223>YNL160W-F
<400>104
ggccattacg gccaaaatga agttccaagt tgttttatc 39
<210>105
<211>21
<212>DNA
<213>Artificial sequence
<220>
<223>YNL160W-R
<400>105
tcatgggaaa atgctttcca g 21
<210>106
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>YGR296C-A-F
<400>106
ggccattacg gccaaaatgg aatctattat cctcagc 37
<210>107
<211>21
<212>DNA
<213>Artificial sequence
<220>
<223>YGR296C-A-R
<400>107
ttaccgtcta gcttccagga g 21
<210>108
<211>39
<212>DNA
<213>Artificial sequence
<220>
<223>YOL154W-F
<400>108
ggccattacg gccaaaatga agttctcttc cggcaaatc 39
<210>109
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YOL154W-R
<400>109
agttacctag acagccacca 20
<210>110
<211>39
<212>DNA
<213>Artificial sequence
<220>
<223>YPL187W-F
<400>110
ggccattacg gccaaaatga gatttccttc aatttttac 39
<210>111
<211>19
<212>DNA
<213>Artificial sequence
<220>
<223>YPL187W-R
<400>111
ttagtacatt ggttggccg 19
<210>112
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>YHR214W-F
<400>112
ggccattacg gccaaaatgt tcaatcgttt taac 34
<210>113
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YHR214W-
<400>113
cggaaacaga accaccgttg 20
<210>114
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>YKR013W-F
<400>114
ggccattacg gccaaaatga aattttctaa agtc 34
<210>115
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YKR013W-R
<400>115
ctcaccaatg acattaccag 20
<210>116
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>YHR139C-F
<400>116
ggccattacg gccaaaatga aattcacatc agtg 34
<210>117
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YHR139C-R
<400>117
gtaactcgct actacttgtg 20
<210>118
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>YIL169C-F
<400>118
ggccattacg gccaaaatgt tcaatcgttt aaac 34
<210>119
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YIL169C-R
<400>119
agttgcgctt gcactagatg 20
<210>120
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>YOL155C-F
<400>120
ggccattacg gccaaaatgt tcaatcgctt taat 34
<210>121
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YOL155C-R
<400>121
agaggcagtg gaagccgatg 20
<210>122
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>YMR325W-F
<400>122
ggccattacg gccaaaatgg tcaaattaac ttca 34
<210>123
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YMR325W-R
<400>123
atagcagtgt agataccgtc 20
<210>124
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>YDR134W-F
<400>124
ggccattacg gccaaaatgc aattctctac cgtc 34
<210>125
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YDR134W-R
<400>125
ttacaacaat aaagcggcag 20
<210>126
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>YLR300W-F
<400>126
ggccattacg gccaaaatgc tttcgcttaa aacg 34
<210>127
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YLR300W-R
<400>127
tgatgatggt cgatagtgac 20
<210>128
<211>30
<212>DNA
<213>Artificial sequence
<220>
<223>SfiA-F
<400>128
ctgagtctca cggccattat ggccaaaatg 30
<210>129
<211>57
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-39-AA
<400>129
Met Arg Leu Ser Asn Leu Ile Ala Ser Ala Ser Leu Leu Ser Ala Ala
1 5 10 15
Thr Leu Ala Ala Pro Ala Asn His Glu His Lys Asp Lys Arg Ala Val
20 25 30
Val Thr Thr Thr Val Gln Lys Gln Thr Thr Val Ile Val Asn Gly Ala
35 40 45
Ala Ser Thr Pro Leu Ala Ala Ser Ala
50 55
<210>130
<211>214
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-39-nt
<400>130
ggccattacg gccaaaatgc gtctctctaa cctaattgct tctgcctctc ttttatctgc 60
tgctactctt gctgcccccg ctaaccacga acacaaggac aagcgtgctg tggtcactac 120
cactgttcaa aaacaaacca ctgtcattgt taatggtgcc gcttcaactc ccctggccgc 180
ctcggcctct gctggcctcg ccttagataa aaga 214
<210>131
<211>128
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-43-AA
<400>131
Met Gln Phe Ser Thr Val Ala Ser Ile Ala Ala Val Ala Ala Val Ala
1 5 10 15
Ser Ala Ala Ala Asn Val Thr Thr Ala Thr Val Ser Gln Glu Ser Thr
20 25 30
Thr Leu Val Thr Ile Thr Ser Cys Glu Asp His Val Cys Ser Glu Thr
35 40 45
Val Ser Pro Ala Leu Val Ser Thr Ala Thr Val Thr Val Asp Asp Val
50 55 60
Ile Thr Gln Tyr Thr Thr Trp Cys Pro Leu Thr Thr Glu Ala Pro Lys
65 70 75 80
Asn Gly Thr Ser Thr Ala Ala pro Val Thr Ser Thr Glu Ala Pro Lys
85 90 95
Asn Thr Thr Ser Ala Ala Pro Thr His Ser Val Thr Ser Tyr Thr Gly
100 105 110
Ala Ala Ala Lys Ala Leu Pro Ala Ala Gly Ala Leu Leu Ala Ala Ser
115 120 125
<210>132
<211>403
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-43-nt
<400>132
ggccattacg gccaaaatgc aattttctac tgtcgcttct atcgccgctg tcgccgctgt 60
cgcttctgcc gctgctaacg ttaccactgc tactgtcagc caagaatcta ccactttggt 120
caccatcact tcttgtgaag accacgtctg ttctgaaact gtctccccag ctttggtttc 180
caccgctacc gtcaccgtcg atgacgttat cactcaatac accacctggt gcccattgac 240
cactgaagcc ccaaagaacg gtacttctac tgctgctcca gttacctcta ctgaagctcc 300
aaagaacacc acctctgctg ctccaactca ctctgtcacc tcttacactg gtgctgctgc 360
taaggctttg ccagctgctg gtgctttgct ggccgcctcg gcc 403
<210>133
<211>71
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-44-AA
<400>133
Met Lys Phe Ser Ser Ala Leu Val Leu Ser Ala Val Ala Ala Thr Ala
1 5 10 15
Leu Ala Glu Ser Ile Thr Thr Thr Ile Thr Ala Thr Lys Asn Gly His
20 25 30
Val Tyr Thr Lys Thr Val Thr Gln Asp Ala Thr Phe Val Trp Gly Gly
35 40 45
Glu Asp Ser Tyr Ala Ser Ser Thr Ser Ala Ala Glu Ser Ser Ala Ala
50 55 60
Glu Thr Ser Ala Ala Ser Ala
65 70
<210>134
<211>229
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-44-nt
<400>134
ggccattacg gccaaaatga aattctcttc cgctttggtt ctatctgctg ttgccgctac 60
tgctcttgct gagagtatca ccaccaccat cactgccacc aagaacggtc atgtctacac 120
taagactgtc acccaagatg ctacttttgt ttggggtggt gaagactctt acgccagcag 180
cacttctgcc gctgaatctt ctgccgccga aacttcggcc gcctcggcc 229
<210>135
<211>119
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-48-AA
<400>135
Met Arg Leu Ser Asn Leu Ile Ala Ser Ala Ser Leu Leu Ser Ala Ala
1 5 10 15
Thr Leu Ala Ala Pro Ala Asn His Glu His Lys Asp Lys Arg Ala Val
20 25 30
Val Thr Thr Thr Val Gln Lys Gln Thr Thr Ile Ile Val Asn Gly Ala
35 40 45
Ala Ser Thr Pro Val Ala Ala Leu Glu Glu Asn Ala Val Val Asn Ser
50 55 60
Ala Pro Ala Ala Ala Thr Ser Thr Thr Ser Ser Ala Ala Ser Val Ala
65 70 75 80
Thr Ala Ala Ala Ser Ser Ser Glu Asn Asn Ser Gln Val Ser Ala Ala
85 90 95
Ala Ser Pro Ala Ser Ser Ser Ala Ala Thr Ser Thr Gln Ser Ser Ser
100 105 110
Ser Ser Leu Ala Ala Ser Ala
115
<210>136
<211>373
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-48-nt
<400>136
ggccattacg gccaaaatgc gtctctctaa cctaattgct tctgcctctc ttttatctgc 60
tgctactctt gctgctcccg ctaaccacga acacaaggac aagcgtgctg tggtcactac 120
cactgttcaa aaacaaacca ctatcattgt taatggtgcc gcttcaactc cagttgctgc 180
tttggaagaa aatgctgttg tcaactccgc tccagctgcc gctaccagta caacatcgtc 240
tgctgcttct gtagctaccg ctgctgcttc ctcttctgag aacaactcac aagtttctgc 300
tgccgcatct ccagcctcca gctctgctgc tacatctact caatcttcct cttcctccct 360
ggccgcctcg gcc 373
<210>137
<211>129
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-52-AA
<400>137
Met Lys Phe Gln Val Val Leu Ser Ala Leu Leu Ala Cys Ser Ser Ala
1 5 10 15
Val Val Ala Ser Pro Ile Glu Asn Leu Phe Lys Tyr Arg Ala Val Lys
20 25 30
Ala Ser His Ser Lys Asn Ile Asn Ser Thr Leu Pro Ala Trp Asn Gly
35 40 45
Ser Asn Ser Ser Asn Val Thr Tyr Ala Asn Gly Thr Asn Ser Thr Thr
50 55 60
Asn Thr Thr Thr Ala Glu Ser Ser Gln Leu Gln Ile Ile Val Thr Gly
65 70 75 80
Gly Gln Val Pro Ile Thr Asn Ser Ser Leu Thr His Thr Asn Tyr Thr
85 90 95
Arg Leu Phe Asn Ser Ser Ser Ala Leu Asn Ile Thr Glu Leu Tyr Asn
100 105 110
Val Ala Arg Val Val Asn Glu Thr Ile Gln Asp Asn Leu Ala Ala Ser
115 120 125
Ala
<210>138
<211>403
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-52-nt
<400>138
ggccattacg gccaaaatga agttccaagt tgttttatct gcccttttgg catgttcatc 60
tgccgtcgtc gcaagcccaa tcgaaaacct attcaaatac agggcagtta aggcatctca 120
cagtaagaat atcaactcca ctttgccggc ctggaatggg tctaactcta gcaatgttac 180
ctacgctaat ggaacaaaca gtactaccaa tactactact gccgaaagca gtcaattaca 240
aatcattgta acaggtggtc aagtaccaat caccaacagt tctttgaccc acacaaacta 300
caccagatta ttcaacagtt cttctgcttt gaacattacc gaattgtaca atgttgcccg 360
tgttgttaac gaaacgatcc aagataacct ggccgcctcg gcc 403
<210>139
<211>124
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-54-aa
<400>139
Met Val Lys Leu Thr Ser Ile Val Ala Gly Val Ala Ala Ile Ala Ala
1 5 10 15
Gly Val Ala Ala Ala Pro Ala Thr Thr Thr Leu Ser Pro Ser Asp Glu
20 25 30
Arg Val Asn Leu Val Glu Leu Gly Val Tyr Val Ser Asp Ile Arg Ala
35 40 45
His Leu Ala Glu Tyr Tyr Met Phe Gln Ala Ala His Pro Thr Glu Thr
50 55 60
Tyr Pro Val Glu Ile Ala Glu Ala Val Phe Asn Tyr Gly Asp Phe Thr
65 70 75 80
Thr Met Leu Thr Gly Ile Pro Ala Asp Gln Val Thr Arg Val Ile Thr
85 90 95
Gly Val Pro Trp Tyr Ser Thr Arg Leu Arg Pro Ala Ile Ser Ser Ala
100 105 110
Leu Ser Lys Asp Gly Ile Tyr Thr Ala Ala Ser Ala
115 120
<210>140
<211>388
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-54-nt
<400>140
ggccattacg gccaaaatgg tcaaactaac ttcaattgtt gctggtgtcg ctgctattgc 60
tgctggtgtc gctgctgccc cagccaccac tactttatct ccctctgatg aaagagttaa 120
cctggtcgaa ttaggtgtct acgtctcaga tatcagagct catttggctg aatactatat 180
gttccaagct gctcatccaa ctgaaactta cccagttgaa attgctgaag ctgttttcaa 240
ctacggtgat ttcaccacta tgttgactgg tattcccgct gatcaegtca ctagagtcat 300
cactggtgtc ccatggtact ccaccagatt gagaccagct atctccagcg ctctatccaa 360
ggacggtatc tacacggccg cctcggcc 388
<210>141
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>YAR066W-F
<400>141
ggccattatg gccaaaatgt tcaatcgttt taaca 35
<210>142
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YAR066W-R
<400>142
gaaccaccgt tgagaatagc 20
<210>143
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>YFR026C-F(
<400>143
ggccattatg gccaaaatga cgccctatgc agtag 35
<210>144
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YFR026C-R
<400>144
tcactttcca gagctataag 20
<210>145
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>YJL158C-F
<400>145
ggccattatg gccaaaatgc aattcaaaaa cgtcg 35
<210>146
<211>21
<212>DNA
<213>Artificial sequence
<220>
<223>YJL158C-R
<400>146
gtcgaccaaa gaaacagctt c 21
<210>147
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>YGR106C-F
<400>147
ggccattatg gccaaaatgg tgttcggtca gctg 34
<210>148
<211>22
<212>DNA
<213>Artificial sequence
<220>
<223>YGR106C-R
<400>148
ccaacgcacc atatgtgata tc 22
<210>149
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>YDR077W-F
<400>149
ggccattatg gccaaaatga aattatcaac tgtcc 35
<210>150
<211>19
<212>DNA
<213>Artificial sequence
<220>
<223>YDR077W-R
<400>150
taacatagca acaccagcc 19
<210>151
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>YIL123W-F
<400>151
ggccattatg gccaaaatga aattctcaac tgccg 35
<210>152
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YIL123W-R
<400>152
acagagacgg tacacccgtc 20
<210>153
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>YNL190W-F
<400>153
ggccattatg gccaaaatga agttctcttc tgttac 36
<210>154
<211>22
<212>DNA
<213>Artificial sequence
<220>
<223>YNL190W-R
<400>154
gcaccggcta cggcagcact ac 22
<210>155
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>YBR078W-F
<400>155
ggccattatg gccaaaatgc aattcaagaa cgctt 35
<210>156
<211>21
<212>DNA
<213>Artificial sequence
<220>
<223>YBR078W-R
<400>156
cagtgatgaa ccaaccgtct c 21
<210>157
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>YJL178C-F
<400>157
ggccattatg gccaaaatgg tatcgaagac ttggat 36
<210>158
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YJL178C-R
<400>158
aacggcgcta taaccgcctc 20
<210>159
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>YMR307W-F
<400>159
ggccattatg gccaaaatgt tgtttaaatc cctttc 36
<210>160
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YMR307W-R
<400>160
gcaaaaccga caccagcggc 20
<210>161
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>YOR247W-F
<400>161
ggccattatg gccaaaatgc ttcaatccgt tgtct 35
<210>162
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YOR247W-R
<400>162
actggtcgaa ttagtaatcg 20
<210>163
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>YJL159W-F
<400>163
ggccattatg gccaaaatgc aatacaaaaa gactttg 37
<210>164
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YJL159W-R
<400>164
aaatcgatag cttccaagtg 20
<210>165
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>YOR085W-F
<400>165
ggccattatg gccaaaatga attggctgtt tttgg 35
<210>166
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YOR085W-R
<400>166
tttgaatggt gccgataacc 20
<210>167
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>YKR042W-F
<400>167
ggccattatg gccaaaatga aattatccgc tctatt 36
<210>168
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YKR042W-R
<400>168
gacaaagtta gcagaaccag 20
<210>169
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>YEL060C-F
<400>169
ggccattatg gccaaaatga agttagaaaa tactc 35
<210>170
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YEL060C-R
<400>170
cttgggtgaa gtaaccgatg 20
<210>171
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>YLR390W-A-F
<400>171
ggccattatg gccaaaatgc gtgccaccac tttatta 37
<210>172
<211>20
<212>DNA
<213>Artificial sequence
<220>
<223>YLR390W-A-R
<400>172
aacatagcgg caacagcagc 20
<210>173
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>YMR251W-A-F
<400>173
ggccattatg gccaaaatga agttatctca agttg 35
<210>174
<211>18
<212>DNA
<213>Artificial sequence
<220>
<223>YMR251W-A-R
<400>174
aatcaaaaag gccaaagc 18
<210>175
<211>99
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-40-aa
<400>175
Met Arg Leu Ser Asn Leu Ile Ala Ser Ala Ser Leu Leu Ser Ala Ala
1 5 10 15
Thr Leu Ala Ala Pro Ala Asn His Glu His Lys Asp Lys Arg Ala Val
20 25 30
Val Thr Thr Thr Val Gln Lys Gln Thr Thr Ile Ile Val Asn Gly Ala
35 40 45
Ala Ser Thr Pro Val Ala Ala Leu Glu Glu Asn Ala Val Val Asn Ser
50 55 60
Ala pro Ala Ala Ala Thr Ser Thr Thr Ser Ser Ala Ala Ser Val Ala
65 70 75 80
Thr Ala Ala Ala Ser Ser Ser Glu Asn Asn Ser Gln Val Ser Val Ala
85 90 95
Ala Ser Ala
<210>176
<211>313
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-40-nt
<400>176
ggccattacg gccaaaatgc gtctctctaa cctaattgct tctgcctctc ttttatctgc 60
tgctactctt gctgctcccg ctaaccacga acacaaggac aagcgtgctg tggtcactac 120
cactgttcaa aaacaaacca ctatcattgt taatggtgcc gcttcaactc cagttgctgc 180
tttggaagaa aatgctgttg tcaactccgc tccagctgcc gctaccagta caacatcgtc 240
tgctgcttct gtagctaccg ctgctgcttc ctcttctgag aacaactcac aagtttctgt 300
ggccgcctcg gcc 313
<210>177
<211>85
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-50-aa
<400>177
Met Leu Gln Ser Val Val Phe Phe Ala Leu Leu Thr Phe Ala Ser Ser
1 5 10 15
Val Ser Ala Ile Tyr Ser Asn Asn Thr Val Ser Thr Thr Thr Thr Leu
20 25 30
Ala pro Ser Tyr Ser Leu Val Pro Gln Glu Thr Thr Ile Ser Tyr Ala
35 40 45
Asp Asp Thr Thr Thr Phe Phe Val Thr Ser Thr Val Tyr Ser Thr Ser
50 55 60
Trp Phe Thr Ser Thr Ser Ala Thr Ile Thr Asn Ala Ala Ser Ser Ser
65 70 75 80
Leu Ala Ala Ser Ala
85
<210>178
<211>271
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-50-nt
<400>178
ggccattacg gccaaaatgc ttcaatccgt tgtctttttc gctcttttaa ccttcgcaag 60
ttctgtgtca gcgatttatt caaacaatac tgtttctaca actaccactt tagcgcccag 120
ctactccttg gtgccccaag agactaccat atcgtacgcc gacgacacca ctaccttttt 180
tgtcacctca acggtctact ccacgagctg gttcacctca acttcagcca ccattaccaa 240
tgcggcctcc tcctccctgg ccgcctcggc c 271
<210>179
<211>116
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-51-aa
<400>179
Met Leu Gln Ser Val Val Phe Phe Ala Leu Leu Thr Phe Ala Ser Ser
1 5 10 15
Val Ser Ala Ile Tyr Ser Asn Asn Thr Val Ser Thr Thr Thr Thr Leu
20 25 30
Ala Pro Ser Tyr Ser Leu Val Pro Gln Glu Thr Thr Ile Ser Tyr Ala
35 40 45
Asp Asp Thr Thr Thr Phe Phe Ala Thr Ser Thr Val Tyr Ser Thr Ser
50 55 60
Trp Phe Thr Ser Thr Ser Ala Thr Ile Thr Asn Ala Ala Ser Ser Ser
65 70 75 80
Leu Ser Thr Ser Ser Ala Ser Gly Ser Val Thr Pro Glu Ser Thr His
85 90 95
Glu Ile Thr Ser Thr Ser Thr Ile Thr Ser Thr Ser Leu Leu Thr Leu
100 105 110
Ala Ala Ser Ala
115
<210>180
<211>364
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-51-nt
<400>180
ggccattacg gccaaaatgc ttcaatccgt tgtctttttc gctcttttaa ccttcgcaag 60
ttctgtgtca gcgatttatt caaacaatac tgtttctaca actaccactt tagcgcccag 120
ctactccttg gtgccccaag agactaccat atcgtacgcc gacgacacca ctaccttttt 180
tgccacctca acggtctact ccacgagctg gttcacctca acttcagcca ccattaccaa 240
tgcggcctcc tcctccttgt ccacctcttc ggcctctgga tctgtaaccc cagaatccac 300
ccatgaaatt acctccacct cgactatcac gtccacttcg ctgctaaccc tggccgcctc 360
ggcc 364
<210>181
<211>114
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-57-aa
<400>181
Met Phe Asn Arg Phe Asn Lys Leu Gln Ala Ala Leu Ala Leu Val Leu
1 5 10 15
Tyr Ser Gln Ser Ala Leu Gly Gln Tyr Tyr Thr Asn Ser Ser Ser Ile
20 25 30
Ala Ser Asn Ser Ser Thr Ala Val Ser Ser Thr Ser Ser Gly Ser Val
35 40 45
Ser Ile Ser Ser Ser Ile Glu Leu Thr Ser Ser Thr Ser Asp Val Ser
50 55 60
Ser Ser Leu Thr Glu Leu Thr Ser Ser Ser Thr Glu Val Ser Ser Ser
65 70 75 80
Ile Ala Pro Ser Thr Ser Ser Ser Glu Val Ser Ser Ser Ile Thr Ser
85 90 95
Ser Gly Ser Ser Val Ser Gly Ser Ser Ser Ile Thr Ser Leu Ala Ala
100 105 110
Ser Ala
<210>182
<211>358
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-57-nt
<400>182
ggccattacg gccaaaatgt tcaatcgctt taataaactt caagccgctt tggctttggt 60
cctttactcc caaagtgcat tgggccaata ttataccaac agttcctcaa tcgctagtaa 120
cagctccacc gccgtttcgt caacttcatc aggttccgtt tccatcagta gttctattga 180
gttgacctca tctacttctg atgtctcgag ctctctcact gagttaacgt catcctccac 240
cgaagtctcg agctccattg ctccatcaac ctcgtcctct gaagtctcga gctctattac 300
ttcatcaggc tcttcagtct ccggctcatc ttctattact tccctggccg cctcggcc 358
<210>183
<211>199
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-58-aa
<400>183
Met Phe Asn Arg Phe Asn Lys Phe Gln Ala Ala Val Ala Leu Ala Leu
1 5 10 15
Leu Ser Arg Gly Ala Leu Gly Asp Ser Tyr Thr Asn Ser Thr Ser Ser
20 25 30
Ala Asp Leu Ser Ser Ile Thr Ser Val Ser Ser Ala Ser Ala Ser Ala
35 40 45
Thr Ala Ser Asp Ser Leu Ser Ser Ser Asp Gly Thr Val Tyr Leu Pro
50 55 60
Ser Thr Thr Ile Ser Gly Asp Leu Thr Val Thr Gly Lys Val Ile Ala
65 70 75 80
Thr Glu Ala Val Glu Val Ala Ala Gly Gly Lys Leu Thr Leu Leu Asp
85 90 95
Gly Glu Lys Tyr Val Phe Ser Ser Asp Leu Lys Val His Gly Asp Leu
100 105 110
Val Val Glu Lys Ser Glu Ala Ser Tyr Glu Gly Thr Ala Phe Asp Val
115 120 125
Ser Gly Glu Thr Phe Glu Val Ser Gly Asn Phe Ser Ala Glu Glu Thr
130 135 140
Gly Ala Val Ser Ala Ser Ile Tyr Ser Phe Thr Pro Ser Ser Phe Lys
145 150 155 160
Ser Ser Gly Asp Ile Ser Leu Ser Leu Ser Lys Ala Lys Lys Gly Glu
165 170 175
Val Thr Phe Ser Pro Tyr Ser Asn Ala Gly Thr Phe Ser Leu Ser Asn
180 185 190
Ala Ile Leu Ala Ala Ser Ala
195
<210>184
<211>613
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-58-nt
<400>184
ggccattacg gccaaaatgt tcaatcgttt taacaaattc caagctgctg tcgctttggc 60
cctactctct cgcggcgctc tcggtgactc ttacaccaat agcacctcct ccgcagactt 120
gagttctatc acttccgtct cgtcagctag tgcaagtgcc accgcttccg actcactttc 180
ttccagtgac ggtaccgttt atttgccatc cacaacaatt agcggtgatc tcacagttac 240
tggtaaagta attgcaaccg aggccgtgga agtcgctgcc ggtggtaagt tgactttact 300
tgacggtgaa aaatacgtct tctcatctga tctaaaagtt cacggtgatt tggttgtcga 360
aaagtctgaa gcaagctacg aaggtaccgc cttcgacgtt tctggtgaga cttttgaagt 420
ttccggtaac ttcagtgctg aagaaactgg cgctgtctcc gcatctatct attcattcac 480
acctagctcg ttcaagagca gcggtgacat ttctttgagt ttgtcaaagg ccaagaaggg 540
tgaagtcacc ttttctccat actctaacgc tggtaccttt tctttgtcaa atgctattct 600
ggccgcctcg gcc 613
<210>185
<211>55
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-59-aa
<400>185
Met Asn Trp Leu Phe Leu Val Ser Leu Val Phe Phe Cys Gly Val Ser
1 5 10 15
Thr His pro Ala Leu Ala Met Ser Ser Asn Arg Leu Leu Lys Leu Ala
20 25 30
Asn Lys Ser Pro Lys Lys Ile Ile Pro Leu Lys Asp Ser Ser Phe Glu
35 40 45
Asn Ile Leu Ala Ala Ser Ala
50 55
<210>186
<211>181
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-59-nt
<400>186
ggccattacg gccaaaatga attggctgtt tttggtctcg ctggttttct tctgcggcgt 60
gtcaacccat cctgccctgg caatgtccag caacagacta ctaaagctgg ctaataaatc 120
tcccaagaaa attatacctc tgaaggactc aagttttgaa aacatcctgg ccgcctcggc 180
c 181
<210>187
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>T1-F
<400>187
ggccattacg gccaaaatgt tcaatcgttt taac 34
<210>188
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>T1-R
<400>188
ttgtagtgtt gactggagca ccgagagcgc cgcgaga 37
<210>189
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>T2-F
<400>189
ggccattacg gccaaaatga cgccctatgc agtag 35
<210>190
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>T2-R
<400>190
ttgtagtgtt gactggagct gcgctcactg ttacaat 37
<210>191
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>T3-F
<400>191
ggccattacg gccaaaatgc aattcaaaaa cgtc 34
<210>192
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>T3-R
<400>192
ttgtagtgtt gactggagca gcagaagcag tggcgga 37
<210>193
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>T4-F
<400>193
ggccattacg gccaaaatga gatttgcaga attc 34
<210>194
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>T4-R
<400>194
ttgtagtgtt gactggagca gccatccccc cgcctaac 38
<210>195
<211>18
<212>DNA
<213>Artificial sequence
<220>
<223>MF-pro-F
<400>195
gctccagtca acactaca 18
<210>196
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>MF-R
<400>196
ggccgaggcg gccgataccc cttcttcttt agcagc 36
<210>197
<211>34
<212>DNA
<213>Artificial sequence
<220>
<223>MF-Pre-F
<400>197
ggccattacg gccaaaatgg tatcgaagac ttgg 34
<210>198
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>KR-IGF-F
<400>198
ctcgccttag ataaaagagg accggagacg ctctgc 36
<210>199
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>IGF-R
<400>199
cactccgttc aagtcgactc aagctgactt ggcagg 36
<210>200
<211>88
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-5-aa
<400>200
Met Phe Asn Arg Phe Asn Lys Phe Gln Ala Ala Val Ala Leu Ala Leu
1 5 10 15
Leu Ser Arg Gly Ala Leu Gly Ala Pro Val Asn Thr Thr Thr Glu Asp
20 25 30
Glu Thr Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu
35 40 45
Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn
50 55 60
Asn Gly Leu Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys
65 70 75 80
Glu Glu Gly Val Ala Ala Ser Ala
85
<210>201
<211>280
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-5-NT
<400>201
ggccattacg gccaaaatgt tcaatcgttt taacaaattc caagctgctg tcgctttggc 60
cctactctct cgcggcgctc tcggtgctcc agtcaacact acaacagaag atgaaacggc 120
acaaattccg gctgaagctg tcatcggtta cttagattta gaaggggatt tcgatgttgc 180
tgttttgcca ttttccaaca gcacaaataa cgggttattg tttataaata ctactattgc 240
cagcattgct gctaaagaag aaggggtggc cgcctcggcc 280
<210>202
<211>84
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-6-aa
<400>202
Met Thr Pro Tyr Ala Val Ala Ile Thr Val Ala Leu Leu Ile Val Thr
1 5 10 15
Val Ser Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ala Ala Ser Ala
<210>203
<211>268
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-6-NT
<400>203
ggccattacg gccaaaatga cgccctatgc agtagcaatt accgtggcct tactaattgt 60
aacagtgagc gcagctccag tcaacactac aacagaagat gaaacggcac aaattccggc 120
tgaagctgtc atcggttact tagatttaga aggggatttc gatgttgctg ttttgccatt 180
ttccaacagc acaaataacg ggttattgtt tataaatact actattgcca gcattgctgc 240
taaagaagaa ggggtggccg cctcggcc 268
<210>204
<211>86
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-7-aa
<400>204
Met Gln Phe Lys Asn Val Ala Leu Ala Ala Ser Val Ala Ala Leu Ser
1 5 10 15
Ala Thr Ala Ser Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr
20 25 30
Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly
35 40 45
Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly
50 55 60
Leu Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu
65 70 75 80
Gly Val Ala Ala Ser Ala
85
<210>205
<211>274
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-7-NT
<400>205
ggccattacg gccaaaatgc aattcaaaaa cgtcgcccta gctgcctccg ttgctgctct 60
atccgccact gcttctgctg ctccagtcaa cactacaaca gaagatgaaa cggcacaaat 120
tccggctgaa gctgtcatcg gttacttaga tttagaaggg gatttcgatg ttgctgtttt 180
gccattttcc aacagcacaa ataacgggtt attgtttata aatactacta ttgccagcat 240
tgctgctaaa gaagaagggg tggccgcctc ggcc 274
<210>206
<211>83
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-8-aa
<400>206
Met Arg Phe Ala Glu Phe Leu Val Val Phe Ala Thr Leu Gly Gly Gly
1 5 10 15
Met Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile
20 25 30
Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe Asp
35 40 45
Val Ala Val Leu Ser Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe
50 55 60
Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ala
65 70 75 80
Ala Ser Ala
<210>207
<211>265
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-8-NT
<400>207
ggccattacg gccaaaatga gatttgcaga attcttggtg gtatttgcca cgttaggcgg 60
ggggatggct gctccagtca acactacaac agaagatgaa acggcacaaa ttccggctga 120
agctgtcatc ggttacttag atttagaagg ggatttcgat gttgctgttt tgtcattttc 180
caacagcaca aataacgggt tattgtttat aaatactact attgccagca ttgctgctaa 240
agaagaaggg gtggccgcct cggcc 265
<210>208
<211>84
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-32-aa
<400>208
Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
1 5 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ala Ala Ser Ala
<210>209
<211>268
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-32-NT
<400>209
ggccattacg gccaaaatga gatttccttc aatttttact gcagttttat tcgcagcatc 60
ctccgcatta gctgctccag tcaacactac aacagaagat gaaacggcac aaattccggc 120
tgaagctgtc atcggttact tagatttaga aggggatttc gatgttgctg ttttgccatt 180
ttccaacagc acaaataacg ggttattgtt tataaatact actattgcca gcattgctgc 240
taaagaagaa ggggtggccg cctcggcc 268
<210>210
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>KR-hP10-F
<400>210
ctcgccttag ataaaagagc tattaagaaa gcccac 36
<210>211
<211>35
<212>DNA
<213>Arti ficial sequence
<220>
<223>hP10-Sal-R
<400>211
ctccgttcaa gtcgacttaa tgtcctggga agagg 35
<210>212
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>KR-IL32g-F
<400>212
ctcgccttag ataaaagaat gtgcttcccg aaggtcc 37
<210>213
<211>36
<212>DNA
<213>Artificial sequence
<220>
<223>IL32g-SalI-R
<400>213
cactccgttc aagtcgactc attttgagga ttgggg 36
<210>214
<211>4
<212>PRT
<213>Artificial sequence
<220>
<223>kex2p protease recognition sequence
<400>214
Leu Asp Lys Arg
1
<210>215
<211>4
<212>PRT
<213>Artificial sequence
<220>
<223>Factor Xa recognition sequence
<400>215
Ile Glu Gly Arg
1
<210>216
<211>4
<212>PRT
<213>Artificial sequence
<220>
<223>subtilisin recognition sequence
<400>216
Ala Ala His Tyr
1
<210>217
<211>7
<212>PRT
<213>Artificial sequence
<220>
<223>tobacco etch virus recognition sequence
<400>217
Glu Asn Leu Tyr Phe Gln Gly
1 5
<210>218
<211>7
<212>PRT
<213>Artificial sequence
<220>
<223>thrombin recognition sequence
<400>218
Glu Asn Leu Tyr Phe Gln Gly
1 5
<210>219
<211>105
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-1-aa
<400>219
Met Phe Asn Arg Phe Asn Lys Phe Gln Ala Ala Val Ala Leu Ala Leu
1 5 10 15
Leu Ser Arg Gly Ala Leu Gly Asp Ser Tyr Thr Asn Ala Thr Ser Ser
20 25 30
Ala Asp Leu Ser Ser Ile Thr Ser Val Ser Ser Ala Ser Ala Ser Ala
35 40 45
Thr Ala Ser Asp Ser Leu Ser Ser Ser Asp Gly Thr Val Tyr Leu Pro
50 55 60
Ser Thr Thr Ile Ser Gly Asp Leu Thr Val Thr Gly Lys Val Ile Ala
65 70 75 80
Thr Glu Ala Val Glu Val Ala Ala Gly Gly Lys Leu Thr Leu Leu Asp
85 90 95
Gly Glu Lys Tyr Val Phe Ser Ser Asp
100 105
<210>220
<211>430
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-1-NT
<400>220
gatcgtcata ttcactcttg ttctcataat agcagtccaa gttttcatct ttgcaagctt 60
tactatttct ttctttttat tggtaaactc tcgcccatta caaaaaaaaa agagatgttc 120
aatcgtttta acaaattcca agctgctgtc gctttggccc tactctctcg cggcgctctc 180
ggtgactctt acaccaatag cacctcctcc gcagacttga gttctatcac ttccgtctcg 240
tcagctagtg caagtgccac cgcttccgac tcactttctt ccagtgacgg taccgtttat 300
ttgccatcca caacaattag cggtgatctc acagttactg gtaaagtaat tgcaaccgag 360
gccgtggaag tcgctgccgg tggtaagttg actttacttg acggtgaaaa atacgtcttc 420
tcatctgatc 430
<210>221
<211>117
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-2-aa
<400>221
Met Thr Pro Tyr Ala Val Ala Ile Thr Val Ala Leu Leu Ile Val Thr
1 5 10 15
Val Ser Ala Leu Gln Val Asn Asn Ser Cys Val Ala Phe Pro Pro Ser
20 25 30
Asn Leu Arg Gly Lys Asn Gly Asp Gly Thr Asn Glu Gln Tyr Ala Thr
35 40 45
Ala Leu Leu Ser Ile Pro Trp Asn Gly Pro Pro Glu Ser Leu Arg Asp
50 55 60
Ile Asn Leu Ile Glu Leu Glu Pro Gln Val Ala Leu Tyr Leu Leu Glu
65 70 75 80
Asn Tyr Ile Asn His Tyr Tyr Asn Thr Thr Arg Asp Asn Lys Cys Pro
85 90 95
Asn Asn His Tyr Leu Met Gly Gly Gln Leu Gly Ser Ser Ser Asp Asn
100 105 110
Arg Ser Leu Asn Asp
115
<210>222
<211>424
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-2-NT
<400>222
gatctcattg gattcaagag aaagaaactc tatactggcg ccaaattagc agtgtcaaat 60
ttcgaaaagg tgatgacgcc ctatgcagta gcaattaccg tggccttact aattgtaaca 120
gtgagcgcac tccaggtcac aattcatgtg tcgcttttcc gccaatcaaa tctcagaggc 180
aaaaatggag acggtactaa tgaacagtat gcaactgcac tactttctat tccctggaat 240
ggacctcctg agtcattgag ggatattaat cttattgaac tcgaaccgca agttgcactc 300
tatttgctcg aaaattatat taaccattac tacaacacca caagagacaa taagtgccct 360
aataaccact acctaatggg agggcagttg ggtagctcat cggataatag gagtttgaac 420
gatc 424
<210>223
<211>104
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-3-aa
<400>223
Met Gln Phe Lys Asn Val Ala Leu Ala Ala Ser Val Ala Ala Leu Ser
1 5 10 15
Ala Thr Ala Ser Ala Glu Gly Tyr Thr Pro Gly Glu Pro Trp Ser Thr
20 25 30
Leu Thr Pro Thr Gly Ser Ile Ser Cys Gly Ala Ala Glu Tyr Thr Thr
35 40 45
Thr Phe Gly Ile Ala Val Gln Ala Ile Thr Ser Ser Lys Ala Lys Arg
50 55 60
Asp Val Ile Ser Gln Ile Gly Asp Gly Gln Val Gln Ala Thr Ser Ala
65 70 75 80
Ala Thr Ala Gln Ala Thr Asp Ser Gln Ala Gln Ala Thr Thr Thr Ala
85 90 95
Thr Pro Thr Ser Ser Glu Lys Ile
100
<210>224
<211>642
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-3-NT
<400>224
gatcccgcct agcccttcca gcttttcttt ttcccctttt gctacggtcg agacacggtc 60
gcccaaaaga aacgggtcag cgtgtactgc gccaaaaaaa ttcgcgccga tttaagctaa 120
acgtccacaa acaaaaacaa aaataagaaa taggttgaca gtgggtgaaa aattctcgaa 180
ggtttcatct ccaaacagtc agtatataag tattcgggaa agagagccaa tctatcttgt 240
ggtgggtcta tcttaacctt ctctttttgg cagtagtaat tgtaaatcaa gacacataaa 300
actatttcac tcgctaaact tacatctaaa atgcaattca aaaacgtcgc cctagctgcc 360
tccgttgctg ctctatccgc cactgcttct gctgaaggtt acactccagg tgaaccatgg 420
tccaccttaa ccccaaccgg ctccatctct tgtggtgctg ccgaatacac taccaccttt 480
ggtattgctg ttcaagctat tacctcttca aaagctaaga gagacgttat ctctcaaatt 540
ggtgacggtc aagtccaagc cacttctgct gctactgctc aagccaccga tagtcaagcc 600
caagctacta ctaccgctac cccaaccagc tccgaaaaga tc 642
<210>225
<211>50
<212>PRT
<213>Artificial sequence
<220>
<223>TFP-4-aa
<400>225
Met Arg Phe Ala Glu Phe Leu Val Val Phe Ala Thr Leu Gly Gly Gly
1 5 10 15
Met Ala Ala Pro Val Glu Ser Leu Ala Gly Thr Gln Arg Tyr Leu Val
20 25 30
Gln Met Lys Glu Arg Phe Thr Thr Glu Lys Leu Cys Ala Leu Asp Asp
35 40 45
Lys Ile
50
<210>226
<211>179
<212>DNA
<213>Artificial sequence
<220>
<223>TFP-4-NT
<400>226
gatccgcttt ttattgcttt gctttgctaa tgagatttgc agaattcttg gtggtatttg 60
ccacgttagg cggggggatg gctgcaccgg ttgagtctct ggccgggacc caacggtatc 120
tggtgcaaat gaaggagcgg ttcaccacag agaagctgtg tgctttggac gacaagatc 179
<210>227
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>PpTFP1-F
<400>227
agtggccatt acggccaaaa tgcaattcaa cagtg 35
<210>228
<211>39
<212>DNA
<213>Artificial sequence
<220>
<223>PpTFP1-R
<400>228
tagggccgag gcggccagtg tggccgatgg gtcccattg 39
<210>229
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>PpTFP2-F
<400>229
agtggccatt acggccaaaa tgcaattctc tatcg 35
<210>230
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>PpTFP2-R
<400>230
tagggccgag gcggccagtg gggtggagtg ggtggttg 38
<210>231
<211>37
<212>DNA
<213>Artificial sequence
<220>
<223>PpTFP3-F
<400>231
agtggccatt acggccaaaa tgaagttttc cactgcg 37
<210>232
<211>38
<212>DNA
<213>Artificial sequence
<220>
<223>PpTFP3-R
<400>232
tagggccgag gcggccaggg tagtggtagg atctggag 38
<210>233
<211>35
<212>DNA
<213>Artificial sequence
<220>
<223>PpTFP4-F
<400>233
agtggccatt acggccaaaa tgcaatacag atctc 35
PCT/RO/134表
申请人或代理人的案卷号: | 国际申请号:TBA |
关于保藏的微生物或其他的生物材料的说明
(PCT 13bis条)
Form PCT/RO/134(July1998)
大肠杆菌
DH5@/pYGT17-IL2
澳大利亚
在此,申请人声明:在本申请授予专利之前,或该申请失效、驳回或撤回之前,只有将微生物样品提供给与本发明利益无关的技术人员才是有效的(澳大利亚专利实施细则第3.25(3)条)。
加拿大
在此,申请人请求:只有基于本申请的加拿大专利被授权或该申请被驳回,或被放弃并不再恢复,或被撤回的情况下,将本申请中涉及的保藏的生物材料样品提供给由专利局长指定的独立的技术人员才是有效的。
丹麦
在此,申请人请求:只有当本申请被公布(由丹麦专利局),或由丹麦专利局最终决定不公布的情况下,将微生物样品提供给本领域技术人员才是有效的(丹麦专利法第22和33(3)部分)。
芬兰
在此,申请人请求:只有在国家专利注册委员会公布授予专利或如果国家专利注册委员会最终决定不授予本申请专利的情况下从提交日之日起的20年起,将微生物样品提供给本领域技术人员才是有效的。
冰岛
在此,申请人请求:只有冰岛专利局授予专利或最终决定不授予本申请专利的情况下,将微生物样品提供给本领域技术人员才是有效的(冰岛专利法第22和33(3)部分)。
荷兰
在此,申请人请求:只有授予荷兰专利之日起或只有该申请被驳回、撤回或失效之日起,根据专利法第31F(1)条的规定只有将微生物样品提供给本领域技术人员才是许可的。
大肠杆菌
DH5@/pYGT17-IL2
挪威
在此,申请人请求:只有当本申请被公布(由挪威专利局),或由挪威专利局最终决定不公布的情况下,将微生物样品提供给本领域技术人员才是有效的(挪威专利法第22和33(3)部分)。
新加坡
在此,申请人请求:微生物样品只能提供给本领域技术人员。
瑞典
在此,申请人请求:只有当本申请被公布(由瑞典专利局),或由瑞典专利局最终决定不
公布的情况下,将微生物样品提供给本领域技术人员才是有效的。
英国
在此,申请人请求:微生物样品只能提供给本领域技术人员。
Claims (119)
1.鉴定靶蛋白特异的翻译融合配偶体(TFP)的方法,所述方法包括:
(i)用多个线性载体和一个编码靶蛋白的核酸共转化多个报告蛋白缺乏的宿主细胞来制备多个被转化的宿主细胞,
其中每个所述线性载体包含一个来自于核酸片段文库中的核酸片段和一个编码N末端氨基酸缺失的报告蛋白的核苷酸序列,和
其中所述编码靶蛋白的核酸在3’末端包含一个编码N末端氨基酸的核苷酸序列,其中在所述线性载体内所述报告蛋白缺乏该N末端氨基酸,并在5’末端包含一个连接DNA;
(ii)在允许所述线性载体和所述编码靶蛋白的核酸体内重组的有效条件下培养所述的多个被转化的宿主细胞;
(iii)从(ii)中的多个被转化的宿主细胞中鉴定出显示报告蛋白活性的细胞;和
(iv)从(iii)中鉴定的细胞中鉴定出TFP;
其中所述TFP包含诱导所述靶蛋白分泌的核酸片段。
2.鉴定靶蛋白特异的TFP文库的方法,所述方法包括:
(i)用多个线性载体和一个编码靶蛋白的核酸共转化多个报告蛋白缺乏的宿主细胞来制备多个被转化的宿主细胞,
其中每个所述的线性载体包含一个来自于核酸片段文库的核酸片段和一个编码N末端氨基酸缺失的报告蛋白的核苷酸序列,和
其中所述编码靶蛋白的核酸在3’末端包含一个编码N末端氨基酸的核苷酸序列,其中在所述线性载体内所述报告蛋白缺乏该N末端氨基酸,并在5’末端包含一个连接DNA;
(ii)在允许所述线性载体和所述编码靶蛋白的核酸体内重组的有效条件下培养多个所述被转化的宿主细胞;
(iii)从(ii)中的多个被转化的宿主细胞中鉴定出显示报告蛋白活性的细胞;和
(iv)从(iii)中鉴定的细胞中鉴定出TFP文库;
其中所述TFP文库包含单独诱导所述靶蛋白分泌的核酸片段。
3.根据权利要求1或2所述的方法,其中所述核酸片段文库来自植物、细菌、酵母、真菌或动物的基因组DNA或cDNA。
4.根据权利要求1或2所述的方法,其中所述核酸片段文库来自重组DNA。
5.根据权利要求3所述的方法,其中所述酵母选自念珠菌属、德巴利酵母属、汉森酵母属、克鲁维酵母属、毕赤酵母属、裂殖酵母属、耶罗威亚酵母属、酵母菌、许旺酵母属和Arxula属。
6.根据权利要求5所述的方法,其中所述酵母选自产朊假丝酵母、博伊丁假丝酵母、白色念珠菌、产乳糖酶酵母、巴斯德毕赤酵母、木糖发酵酵母、粟酒裂殖酵母、酿酒酵母、多形汉森酵母、解脂耶氏酵母、西方许旺酵母和Arxula adeninivorans。
7.根据权利要求3所述的方法,其中所述真菌选自曲霉属、青霉属、根霉属和木霉属。
8.根据权利要求3所述的方法,其中所述细菌选自埃希菌属、假单胞杆菌属和杆菌属。
9.根据权利要求3所述的方法,其中所述植物选自拟南芥、玉米、烟草和马铃薯。
10.根据权利要求3所述的方法,其中所述动物选自人、小鼠、大鼠、兔、狗、猫和猴。
11.根据权利要求1-10中任一权利要求所述的方法,其中所述核酸片段文库是预先选定的候选TFPs文库。
12.根据权利要求11所述的方法,其中所述的预先选定的候选TFPs文库是通过用包含核酸片段文库和编码报告蛋白的核酸的多个载体转化多个报告蛋白缺乏的宿主细胞、收集生长的细胞、从该细胞中分离出载体,和从该载体中分离出核酸片段,从而获得包含单独诱导报告蛋白分泌的核酸片段的TFP文库来获得。
13.根据权利要求11所述的方法,其中所述预先选定的候选TFPs文库来源于在基因组数据库中经鉴定的序列,该序列通过搜索(i)含有与那些一个或多个预先鉴定了的TFPs同源的前分泌信号的基因;(ii)包含分泌信号序列的基因;或(iii)编码穿过内质网的蛋白质的基因而获得。
14.根据权利要求11所述的方法,其中所述的预先选定的候选TFPs文库是通过改变先前鉴定了的TFPs获得的。
15.根据权利要求11所述的方法,其中所述的预先选定的候选TFPs文库是通过人工设计核酸片段获得的,其中所述的核酸片段的前和原信号序列在先前鉴定了的TFPs之间进行互换。
16.根据权利要求11所述的方法,其中所述的预先选定的候选TFPs文库是核心TFPs文库,其中所述的核心TFPs是先前鉴定对一个或多个靶蛋白有效的TFPs的集合。
17.根据权利要求1-16中任一权利要求所述的方法,其中所述的核酸片段大小少于1000个碱基对。
18.根据权利要求17所述的方法,其中所述核酸片段大小少于700个碱基对。
19.根据权利要求18所述的方法,其中所述核酸片段大小少于500个碱基对。
20.根据权利要求19所述的方法,其中所述核酸片段大小少于300个碱基对。
21.根据权利要求1-20中任一权利要求所述的方法,其中所述核酸片段文库是通过DNA的酶断裂构建的。
22.根据权利要求1-20中任一权利要求所述的方法,其中所述核酸片段文库是通过cDNA合成构建的。
23.根据权利要求1-20中任一权利要求所述的方法,其中所述核酸片段文库是通过重组DNA技术构建的。
24.根据权利要求23所述的方法,其中所述重组DNA技术包含单向缺失。
25.根据权利要求1-24中任一权利要求所述的方法,其中所述宿主细胞选自植物、细菌、真菌、酵母或动物细胞。
26.根据权利要求25所述的方法,其中所述酵母选自念珠菌属、德巴利酵母属、汉森酵母属、克鲁维酵母属、毕赤酵母属、裂殖酵母属、耶罗威亚酵母属、酵母菌、许旺酵母属和Arxula属。
27.根据权利要求26所述的方法,其中所述酵母选自产朊假丝酵母、博伊丁假丝酵母、白色念珠菌、产乳糖酶酵母、巴斯德毕赤酵母、木糖发酵酵母、粟酒裂殖酵母、酿酒酵母、多形汉森酵母、解脂耶氏酵母、西方许旺酵母和Arxula adeninivorans。
28.根据权利要求25所述的方法,其中所述真菌选自选自曲霉属、青霉属、根霉属和木霉属。
29.根据权利要求25所述的方法,其中所述细菌选自埃希菌属、假单胞杆菌属和杆菌属。
30.根据权利要求25所述的方法,其中所述植物选自拟南芥、玉米、烟草和马铃薯。
31.根据权利要求25所述的方法,其中所述动物选自人、小鼠、大鼠、兔、狗、猫、猴和昆虫。
32.根据权利要求25所述的方法,其中所述动物细胞选自CHO、COS 1、COS 7、BSC 1、BSC 40、BMT 10和Sf9。
33.根据权利要求1-27中任一权利要求所述的方法,其中所述宿主细胞是酵母细胞,且所述核酸片段是从酵母的基因组或cDNA中分离出来的。
34.根据权利要求1-33中任一权利要求所述的方法,其中所述报告蛋白是分泌至细胞外间隙的蛋白质。
35.根据权利要求34所述的方法,其中所述报告蛋白选自转化酶、蔗糖酶、纤维素酶、木聚糖酶、麦芽糖酶、淀粉酶、葡萄糖淀粉酶、半乳糖苷酶、磷酸酶、β-内酰胺酶、脂肪酶或蛋白酶。
36.根据权利要求35所述的方法,其中所述半乳糖苷酶选自α-半乳糖苷酶、β-半乳糖苷酶和蜜二糖酶。
37.根据权利要求36所述的方法,其中所述报告蛋白是蜜二糖酶。
38.根据权利要求35所述的方法,其中所述磷酸酶是PHO5。
39.根据权利要求35所述的方法,其中所述宿主细胞是酵母,所述报告蛋白是转化酶且所述被转化的酵母细胞是根据它们在蔗糖或棉子糖上生长的能力来选择的。
40.根据权利要求35所述的方法,其中所述宿主细胞是酵母,所述报告蛋白是淀粉酶,所述酵母细胞是非淀粉分解酵母细胞,且所述被转化细胞是通过其降解淀粉的能力来筛选的。
41.根据权利要求1-33中任一权利要求所述的方法,其中所述鉴定细胞显示报告蛋白活性的步骤是通过使用对生长抑制因子具有抗性的报告蛋白来进行的。
42.根据权利要求1-41中任一权利要求所述的方法,其中所述鉴定细胞显示报告蛋白活性的步骤是通过使用两个或多个报告蛋白来进行的。
43.根据权利要求42所述的方法,其中所述鉴定细胞显示报告蛋白活性的步骤是通过使用两个报告蛋白来进行的。
44.根据权利要求43所述的方法,其中所述两个报告蛋白是脂肪酶和转化酶。
45.根据权利要求1-44中任一权利要求所述的方法,其中所述靶蛋白来自植物、动物或微生物。
46.根据权利要求45所述的方法,其中所述靶蛋白是人类蛋白质。
47.根据权利要求45所述的方法,其中所述靶蛋白是细胞因子、血清蛋白、集落刺激因子、生长因子、激素或酶。
48.根据权利要求45所述的方法,其中所述靶蛋白选自白细胞介素、凝结因子、干扰素-α、-β或-γ、粒细胞集落刺激因子、人粒细胞巨噬细胞集落刺激因子、组织生长因子、上皮生长因子、TGFα、TGFβ、表皮生长因子、血小板衍生生长因子、成纤维细胞生长因子、促卵泡激素、促甲状腺激素、血管升压素、色素性激素、甲状旁腺激素、促黄体生成激素释放激素、碳水化合物特异性酶、蛋白水解酶、脂肪酶、氧化还原酶、转移酶、水解酶、裂解酶、异构酶、连接酶、免疫球蛋白、细胞因子受体、乳铁蛋白、磷脂酶A2激活蛋白、胰岛素、肿瘤坏死因子、降钙素、降钙素基因相关肽、脑啡肽、生长调节素、促红细胞生成素、下丘脑释放因子、催乳素、绒毛膜促性腺激素、组织纤溶酶原激活物、生长激素释放肽、胸腺体液因子、抗癌肽或抗菌肽。
49.根据权利要求45所述的方法,其中所述靶蛋白选自人白细胞介素-2、人白细胞介素-1β、人白细胞介素-6、人白细胞介素-32α、-32β或-32γ、VII因子、VIII因子、IX因子、人血清白蛋白、人干扰素-α、-β或-γ、人粒细胞集落刺激因子、人粒细胞巨噬细胞集落刺激因子、人生长激素、人血小板衍生生长因子、人碱性成纤维细胞生长因子、人表皮生长因子、人胰岛素样生长因子、人神经生长因子、人转化生长因子β-1、人促卵泡激素、葡萄糖氧化酶、glucodase、半乳糖苷酶、葡糖脑苷脂酶、葡糖醛酸糖苷酶、门冬酰胺酶、精氨酸酶、精氨酸脱氨基酶、过氧化物歧化酶、endotoxinase、过氧化氢酶、糜蛋白酶、尿酸酶、腺苷二磷酸酶、酪氨酸酶、胆红素氧化酶、牛1-磷酸半乳糖尿苷酸转移酶、水母绿色荧光蛋白、南极假丝酵母脂肪酶B、假丝酵母脂肪酶、真菌氯过氧化物酶、β-半乳糖苷酶、解离酶、α-半乳糖苷酶、β-葡萄糖苷酶、海藻糖合酶、环糊精糖基转移酶、木聚糖酶、植酸酶、人乳铁蛋白、人促红细胞生成素、人对氧磷酶、人生长分化因子15、人半乳凝素-3结合蛋白、人丝氨酸蛋白酶抑制剂、Kunitz2型、人Janus激酶2、人FMS样酪氨酸激酶3配体、人YM1&2、人CEMI、人二酰基甘油酰基转移酶、人瘦蛋白、人mL259、人蛋白水解酶3、人溶菌酶、人DEAD盒蛋白41、人依托泊苷诱导蛋白24、鼠细胞凋亡蛋白酶1、牛血管生成因子和蚯蚓蚓激酶。
50.根据权利要求45所述的方法,其中所述靶蛋白是使用常规重组生产方法难以制备的蛋白质。
51.根据权利要求1-50中任一权利要求所述的方法,其中所述连接DNA长度大于20个碱基对。
52.根据权利要求51所述的方法,其中所述连接DNA长度大于30个碱基对。
53.根据权利要求52所述的方法,其中所述连接DNA长度大于40个碱基对。
54.根据权利要求1-53中任一权利要求所述的方法,其中所述连接DNA编码一个蛋白水解酶识别序列从而允许在TFP和靶蛋白的连接处断裂。
55.根据权利要求54所述的方法,其中所述连接DNA编码酵母kex2p识别序列。
56.根据权利要求55所述的方法,其中所述连接DNA编码包含Lys-Arg或Arg-Arg的氨基酸序列。
57.根据权利要求56所述的方法,其中所述连接DNA编码包含Leu-Asp-Lys-Arg(SEQ ID NO:214)的氨基酸序列。
58.根据权利要求54所述的方法,其中所述连接DNA编码哺乳动物弗林蛋白酶识别序列。
59.根据权利要求58所述的方法,其中所述连接DNA编码包含Arg-X-X-Arg的氨基酸序列。
60.根据权利要求54所述的方法,其中所述连接DNA编码Xa因子识别序列。
61.根据权利要求59所述的方法,其中所述连接DNA编码包含Ile-Glu-Gly-Arg(SEQ ID NO:215)的氨基酸序列。
62.根据权利要求54所述的方法,其中所述连接DNA编码肠激酶识别序列。
63.根据权利要求62所述的方法,其中所述连接DNA编码包含Asp-Asp-Lys的氨基酸序列。
64.根据权利要求54所述的方法,其中所述连接DNA编码枯草杆菌蛋白酶识别序列。
65.根据权利要求64所述的方法,其中所述连接DNA编码包含Ala-Ala-His-Tyr(SEQ ID NO:216)的氨基酸序列。
66.根据权利要求54所述的方法,其中所述连接DNA编码烟草蚀纹病毒蛋白水解酶识别序列。
67.根据权利要求66所述的方法,其中所述连接DNA编码包含Glu-Asn-Leu-Tyr-Phe-Gln-Gly(SEQ ID NO:217)的氨基酸序列。
68.根据权利要求54所述的方法,其中所述连接DNA编码凝血酶识别序列。
69.根据权利要求68所述的方法,其中所述连接DNA编码包含Arg-Gly-Pro-Arg(SEQ ID NO:218)的氨基酸序列。
70.根据权利要求54所述的方法,其中所述连接DNA编码遍在蛋白质水解酶识别序列。
71.根据权利要求70所述的方法,其中所述连接DNA编码包含Arg-Gly-Gly的氨基酸序列。
72.根据权利要求1-71中任一权利要求所述的方法,其中所述连接DNA编码亲和标记物。
73.根据权利要求72所述的方法,其中所述亲和标记物选自GST、MBP、NusA、硫氧还蛋白、遍在蛋白质、FLAG、BAP、6HIS、STREP、CBP、CBD和S标记物。
74.根据权利要求1-73中任一权利要求所述的方法,其中所述连接DNA编码限制性酶识别位点。
75.根据权利要求74所述的方法,其中所述限制性酶识别位点用于SfiI。
76.根据权利要求75所述的方法,其中所述连接DNA还编码kex2p样蛋白水解酶或kex2p识别序列。
77.由权利要求1-76中任一权利要求所述的方法鉴定的TFP或其片段或衍生物。
78.根据权利要求77所述的TFP,其中所述TFP选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ ID NO:33)、TFP-18(SEQ ID NO:35)、TFP-19(SEQID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQ ID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQ ID NO:61)、TFP-22(SEQ ID NO:63)、TFP-29(SEQID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQ ID NO:69)、TFP-39(SEQ ID NO:129)、TFP-43(SEQ ID NO:131)、TFP-44(SEQ ID NO:133)、TFP-48(SEQ ID NO:135)、TFP-52(SEQ ID NO:137)、TFP-54(SEQ ID NO:139)、TFP-40(SEQ ID NO:175)、TFP-50(SEQ ID NO:177)、TFP-51(SEQ ID NO:179)、TFP-57(SEQ ID NO:181)、TFP-58(SEQ ID NO:183)、TFP-59(SEQ ID NO:185)、TFP-5(SEQ ID NO:200)、TFP-6(SEQID NO:202)、TFP-7(SEQ ID NO:204)、TFP-8(SEQ ID NO:206)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ ID NO:86)、PpTFP-3(SEQ ID NO:88)和PpTFP-4(SEQ ID NO:90)组成的组或其衍生物或片段。
79.TFP文库,所述TFP文库包含一个或多个由权利要求1-76中任一权利要求所述的方法鉴定的TFPs或其片段或衍生物。
80.根据权利要求76所述的TFP文库,其中所述TFP文库包含两个或多个TFPs,所述的TFPs选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ IDNO:33)、TFP-18(SEQ ID NO:35)、TFP-19(SEQ ID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQ ID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQID NO:61)、TFP-22(SEQ ID NO:63)、TFP-29(SEQ ID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQ ID NO:69)、TFP-39(SEQ ID NO:129)、TFP-43(SEQ ID NO:131)、TFP-44(SEQ ID NO:133)、TFP-48(SEQ ID NO:135)、TFP-52(SEQ ID NO:137)、TFP-54(SEQ ID NO:139)、TFP-40(SEQ ID NO:175)、TFP-50(SEQ ID NO:177)、TFP-51(SEQ ID NO:179)、TFP-57(SEQ ID NO:181)、TFP-58(SEQ ID NO:183)、TFP-59(SEQ ID NO:185)、TFP-5(SEQ ID NO:200)、TFP-6(SEQ ID NO:202)、TFP-7(SEQID NO:204)、TFP-8(SEQ ID NO:206)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ IDNO:86)、PpTFP-3(SEQ ID NO:88)和PpTFP-4(SEQ ID NO:90)组成的组或其片段或衍生物。
81.根据权利要求80所述的TFP文库,其中所述TFP文库包含四个或更多个所列的TFPs或其片段或衍生物。
82.根据权利要求80所述的TFP文库,其中所述TFP文库包含六个或更多所列TFPs或其片段或衍生物。
83.根据权利要求82所述的TFP文库,其中所述TFP文库包含八个或更多所列TFPs或其片段或衍生物。
84.根据权利要求83所述的TFP文库,其中所述TFP文库包含十个或更多所列TFPs或其片段或衍生物。
85.根据权利要求84所述的TFP文库,其中所述TFP文库包含十二个或更多所列TFPs或其片段或衍生物。
86.根据权利要求79所述的TFP文库,其中所述TFP文库包含六个或更多TFPs,所述TFPs选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ IDNO:33)、TFP-18(SEQ ID NO:35)、TFP-19(SEQ ID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQ ID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQID NO:61)、TFP-22(SEQ ID NO:63)、TFP-29(SEQ ID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQ ID NO:69)、TFP-39(SEQ ID NO:129)、TFP-43(SEQ ID NO:131)、TFP-44(SEQ ID NO:133)、TFP-48(SEQ ID NO:135)、TFP-52(SEQ ID NO:137)、TFP-54(SEQ ID NO:139)、TFP-40(SEQ ID NO:175)、TFP-50(SEQ ID NO:177)、TFP-51(SEQ ID NO:179)、TFP-57(SEQ ID NO:181)、TFP-58(SEQ ID NO:183)、TFP-59(SEQ ID NO:185)、TFP-5(SEQ ID NO:200)、TFP-6(SEQ ID NO:202)、TFP-7(SEQID NO:204)、TFP-8(SEQ ID NO:206)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ IDNO:86)、PpTFP-3(SEQ ID NO:88)、PpTFP-4(SEQ ID NO:90)、TFP-1(SEQ ID NO:219)、TFP-2(SEQ ID NO:221)、TFP-3(SEQ ID NO:223)、TFP-4(SEQ ID NO:225)和TFP 32(SEQID NO:208)组成的组或其片段或衍生物。
87.根据权利要求86所述的TFP文库,其中所述TFP文库包含八个或更多所列TFPs或其片段或衍生物。
88.根据权利要求87所述的TFP文库,其中所述TFP文库包含十个或更多所列TFPs或其片段或衍生物。
89.根据权利要求88所述的TFP文库,其中所述TFP文库包含十二个或更多所列TFPs或其片段或衍生物。
90.根据权利要求89所述的TFP文库,其中所述TFP文库包含十五个或更多所列TFPs或其片段或衍生物。
91.核酸片段文库,其中所述的核酸片段文库包含10个或更多由权利要求12所述的方法鉴定的核酸片段。
92.根据权利要求91所述的核酸片段文库,其中所述的核酸片段文库包含50个或更多由权利要求12所述的方法鉴定的核酸片段。
93.根据权利要求92所述的核酸片段文库,其中所述的核酸片段文库包含100个或更多由权利要求12所述的方法鉴定的核酸片段。
94.根据权利要求93所述的核酸片段文库,其中所述的核酸片段文库包含500个或更多由权利要求12所述的方法鉴定的核酸片段。
95.根据权利要求94所述的核酸片段文库,其中所述的核酸片段文库包含1000个或更多由权利要求12所述的方法鉴定的核酸片段。
96.根据权利要求95所述的核酸片段文库,其中所述的核酸片段文库包含2000个或更多由权利要求12所述的方法鉴定的核酸片段。
97.核酸片段文库,其中所述的核酸片段文库包含10个或更多由权利要求13所述的方法鉴定的核酸片段。
98.根据权利要求97所述的核酸片段文库,其中所述的核酸片段文库包含50个或更多由权利要求13所述的方法鉴定的核酸片段。
99.根据权利要求98所述的核酸片段文库,其中所述的核酸片段文库包含100个或更多由权利要求13所述的方法鉴定的核酸片段。
100.核酸片段文库,其中所述的核酸片段文库包含10个或更多由权利要求14所述的方法鉴定的核酸片段。
101.根据权利要求100所述的核酸片段文库,其中所述的核酸片段文库包含50个或更多由权利要求14所述的方法鉴定的核酸片段。
102.根据权利要求101所述的核酸片段文库,其中所述的核酸片段文库包含100个或更多由权利要求14所述的方法鉴定的核酸片段。
103.根据权利要求102所述的核酸片段文库,其中所述的核酸片段文库包含500个或更多由权利要求14所述的方法鉴定的核酸片段。
104.根据权利要求103所述的核酸片段文库,其中所述的核酸片段文库包含1000个或更多由权利要求14所述的方法鉴定的核酸片段。
105.核酸片段文库,其中所述的核酸片段文库包含10个或更多由权利要求15所述的方法鉴定的核酸片段。
106.根据权利要求105所述的核酸片段文库,其中所述的核酸片段文库包含50个或更多由权利要求15所述的方法鉴定的核酸片段。
107.根据权利要求106所述的核酸片段文库,其中所述的核酸片段文库包含100个或更多由权利要求15所述的方法鉴定的核酸片段。
108.根据权利要求107所述的核酸片段文库,其中所述的核酸片段文库包含500个或更多由权利要求15所述的方法鉴定的核酸片段。
109.包含编码TFP或其片段或衍生物的核苷酸序列和编码靶蛋白的核酸序列的核酸,其中所述TFP选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQID NO:33)、TFP-18(SEQ ID NO:35)、TFP-19(SEQ ID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQ ID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQID NO:61)、TFP-22(SEQ ID NO:63)、TFP-29(SEQ ID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQ ID NO:69)、TFP-39(SEQ ID NO:129)、TFP-43(SEQ ID NO:131)、TFP-44(SEQ ID NO:133)、TFP-48(SEQ ID NO:135)、TFP-52(SEQ ID NO:137)、TFP-54(SEQ ID NO:139)、TFP-40(SEQ ID NO:175)、TFP-50(SEQ ID NO:177)、TFP-51(SEQ ID NO:179)、TFP-57(SEQ ID NO:181)、TFP-58(SEQ ID NO:183)、TFP-59(SEQ ID NO:185)、TFP-5(SEQ ID NO:200)、TFP-6(SEQ ID NO:202)、TFP-7(SEQID NO:204)、TFP-8(SEQ ID NO:206)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ IDNO:86)、PpTFP-3(SEQ ID NO:88)和PpTFP-4(SEQ ID NO:90)组成的组。
110.根据权利要求109所述的核酸,其中所述靶蛋白选自IL-2、IL-32、人生长激素和人胱天蛋白酶-1亚基P10。
111.根据权利要求109所述的核酸,其中所述TFP选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ ID NO:33)、TFP-18(SEQ ID NO:35)、TFP-19(SEQID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQ ID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、PpTFP-1(SEQ ID NO:84)、PpTFP-2(SEQ ID NO:86)、PpTFP-3(SEQ ID NO:88)、PpTFP-4(SEQ ID NO:90)组成的组且所述靶蛋白是IL-2。
112.根据权利要求109所述的核酸,其中所述TFP选自由TFP-11(SEQ ID NO:61)、TFP-22(SEQ ID NO:63)、TFP-29(SEQ ID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQID NO:69)组成的组且所述靶蛋白是IL-32α。
113.根据权利要求109所述的核酸,其中所述TFP选自由TFP-9(SEQ ID NO:29)、TFP-13(SEQ ID NO:31)、TFP-17(SEQ ID NO:33)、TFP-18(SEQ ID NO:35)、TFP-19(SEQID NO:37)、TFP-20(SEQ ID NO:39)、TFP-21(SEQ ID NO:41)、TFP-25(SEQ ID NO:43)、TFP-27(SEQ ID NO:45)、TFP-11(SEQ ID NO:61)、TFP-22(SEQ ID NO:63)、TFP-29(SEQID NO:65)、TFP-34(SEQ ID NO:67)、TFP-38(SEQ ID NO:69)组成的组,且所述靶蛋白是生长激素。
114.制备靶蛋白的方法,其中包含制备载体,所述载体包含编码所述靶蛋白的核苷酸序列,所述核苷酸序列可操作性的连接于编码由权利要求1-76中任一方法鉴定的TFP或其片段或衍生物的核苷酸序列,用所述载体转化宿主细胞,和在产生所述靶蛋白的条件下培养所述宿主细胞。
115.根据权利要求114所述的方法,其中所述载体包含权利要求107所述的核酸。
116.根据权利要求115所述的方法,其中所述靶蛋白选自IL-2、IL-32、人生长激素和人胱天蛋白酶-1亚基P10。
117.线性载体,其中所述线性载体包含一个来自于核酸片段文库的核酸片段和一个编码N末端氨基酸缺失的报告蛋白的核苷酸序列。
118.根据权利要求117所述的线性载体,其中还包含一个编码靶蛋白的核苷酸序列。
119.用多个线性载体和一个编码靶蛋白的核酸转化的多个报告蛋白缺失的宿主细胞,
其中每个所述线性载体包含一个来自于核酸片段文库的核酸片段和一个编码N末端氨基酸缺失的报告蛋白的核苷酸序列,和
其中所述编码靶蛋白的核酸在3’末端包含一个编码N末端氨基酸的核苷酸序列,其中在所述线性载体内所述报告蛋白缺乏所述的N末端氨基酸,并在5’末端包含一个连接DNA。
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CN107849737B (zh) * | 2015-07-03 | 2020-06-26 | 湖南中晟全肽生化有限公司 | 肽文库构建方法及相关载体 |
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Also Published As
Publication number | Publication date |
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US20090181425A1 (en) | 2009-07-16 |
JP2012165748A (ja) | 2012-09-06 |
JP2009501020A (ja) | 2009-01-15 |
JP5662370B2 (ja) | 2015-01-28 |
KR100975596B1 (ko) | 2010-08-13 |
EP1904656B1 (en) | 2016-05-04 |
EP1904656A2 (en) | 2008-04-02 |
KR20080042823A (ko) | 2008-05-15 |
WO2007015178A2 (en) | 2007-02-08 |
WO2007015178A3 (en) | 2007-07-12 |
US8865629B2 (en) | 2014-10-21 |
CN101310023B (zh) | 2015-07-29 |
KR20070009269A (ko) | 2007-01-18 |
EP1904656A4 (en) | 2009-05-06 |
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