CN109563531A - 重组蛋白的生产方法 - Google Patents
重组蛋白的生产方法 Download PDFInfo
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- CN109563531A CN109563531A CN201780047981.7A CN201780047981A CN109563531A CN 109563531 A CN109563531 A CN 109563531A CN 201780047981 A CN201780047981 A CN 201780047981A CN 109563531 A CN109563531 A CN 109563531A
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Abstract
本发明涉及一种重组蛋白的生产方法,其为使用在诱导型启动子的调控下表达重组蛋白的重组细胞的该重组蛋白的生产方法,其中,包括在使重组细胞增殖后的培养液的一部分中添加新鲜的培养基并连续地进行培养的步骤,不重复使用诱导了重组蛋白的表达的重组细胞。
Description
技术领域
本发明涉及利用微生物进行的工业规模下的重组蛋白的生产方法。
背景技术
在重组蛋白的工业规模下的生产中采用流加培养、半连续培养和连续培养等培养方法。
流加培养也称为半分批培养。分批培养是在培养开始时预先将培养所需要的基质(营养源、培养基成分等)全部添加到培养基中的培养方法。与此相对,流加培养是在制备成适度的基质浓度的培养基中开始培养,然后逐次添加被消耗掉的各基质来进行补充的培养方法。分批培养和流加培养都是在培养结束之前不从培养槽(生物反应器)中去除培养液的培养方法。在基质以高浓度存在时会产生增殖抑制的培养系统中,流加培养能够通过将基质浓度控制为适当的低浓度而避免增殖抑制。还有通过流加培养能够进行高细胞密度培养的情况,能够高浓度地生产重组蛋白。作为流加基质的添加方法,已知有分批地逐次添加流加基质的方法、以恒定的流量添加流加基质的恒流量添加法(恒速流加法)、使流加基质的流量按指数函数增加而进行添加的指数流加法等。
连续培养是在保持培养液量恒定的同时,连续地进行新鲜培养基的流加和培养液的排出,在培养液中的培养基组成不随时间变化的稳态下进行培养的培养方法。连续培养中的细胞密度与流加培养中的细胞密度相比通常较低。另一方面,通过连续培养,能够持续地生产重组蛋白。
半连续培养是通过分批培养或流加培养得到特定的容量或生物质后,将包含重组蛋白的培养液的一部分从生物反应器中去除,同时向生物反应器中添加新鲜的培养基,由此持续重复进行重组蛋白的生产的方法(专利文献1)。
对于具有极大的弹性和回弹性、同时具有橡胶样的性质的节肢弹性蛋白和弹性蛋白、作为羊绒和羊毛的主要成分蛋白之一的角蛋白、为了对各种结缔组织赋予力学强度而发挥作用的胶原蛋白、轻柔且结实且具有独特光泽的蚕丝以及具有优良的强度和伸长率的蛛丝等结构蛋白,研究了来源于这些结构蛋白的重组蛋白的工业规模下的生产方法,但现状是,在实用化中还存在很多课题。即使对于全力进行了研究的蛛丝蛋白,生产水平也仍然较低,认为在细菌宿主中表达天然型蛛丝蛋白是低效的(非专利文献1、专利文献2)。
现有技术文献
专利文献
专利文献1:日本特表2010-527239号公报
专利文献2:国际公开第2015/042164号
非专利文献
非专利文献1:Appl Microbiol Biotechnol.,1998年,49(1),pp.31-38.
发明内容
发明所要解决的问题
本发明的目的在于提供以工业规模高效地稳定生产重组蛋白的方法。
用于解决问题的方法
本发明人在对利用经转化的微生物通过连续培养进行重组蛋白的工业规模下的生产方法进行研究的过程中发现,通过不重复使用曾进行了表达诱导的该微生物,能够高效、稳定地高生产目的重组蛋白,从而完成了本发明。
即,本发明涉及例如以下的各发明。
[1]一种重组蛋白的生产方法,其为使用在诱导型启动子的调控下表达重组蛋白的重组细胞的该重组蛋白的生产方法,其中,
包括在使上述重组细胞增殖后的培养液的一部分中添加新鲜的培养基并连续地进行培养的步骤,
不重复使用诱导了上述重组蛋白的表达的重组细胞。
[2]如[1]所述的重组蛋白的生产方法,其中,上述重组细胞的增殖和上述重组蛋白的表达的诱导在不同的培养槽中进行。
[3]如[1]或[2]所述的重组蛋白的生产方法,其中,上述重组细胞的增殖通过流加培养进行。
[4]一种重组蛋白的生产方法,其为使用在诱导型启动子的调控下表达重组蛋白的重组细胞的该重组蛋白的生产方法,其中,
包括重复进行下述(A)~(E)步骤:
(A)通过分批培养或流加培养使上述重组细胞在培养槽中增殖的步骤;
(B)在(A)步骤中的增殖后,将上述培养槽的培养液的一部分转移至接收用培养槽中的步骤;
(C)在(B)步骤中的转移后,在两个培养槽中的任意一个中添加新鲜的培养基,进入(A)步骤的步骤;
(D)在(C)步骤中未进入(A)步骤的另一个培养槽中诱导上述重组蛋白的表达,使上述重组蛋白蓄积的步骤;
(E)从培养液中分离和纯化(D)步骤中蓄积的上述重组蛋白的步骤。
[5]如[1]~[4]中任一项所述的重组蛋白的生产方法,其中,在上述重组细胞的增殖达到对数生长期的中期时,开始上述重组蛋白的表达的诱导。
[6]如[1]~[5]中任一项所述的重组蛋白的生产方法,其中,上述重组蛋白的表达的诱导通过在培养液中添加表达诱导剂、或者改变培养液的温度来进行。
[7]如[4]所述的重组蛋白的生产方法,其中,以培养液总量为基准,在(B)步骤中转移至接收用培养槽中的培养液的量为80~99体积%。
[8]如[1]~[7]中任一项所述的重组蛋白的生产方法,其中,上述重组细胞为编码上述重组蛋白的基因被整合至染色体中的细胞。
[9]如[1]~[8]中任一项所述的重组蛋白的生产方法,其中,上述重组蛋白为结构蛋白。
[10]如[9]所述的重组蛋白的生产方法,其中,上述结构蛋白为来源于选自由角蛋白、胶原蛋白、弹性蛋白、节肢弹性蛋白、蚕丝和蛛丝组成的组中的蛋白质的蛋白质。
[11]如[1]~[10]中任一项所述的重组蛋白的生产方法,其中,上述诱导型启动子为选自T7启动子、tac启动子、trc启动子、lac启动子和lacUV5启动子中的IPTG诱导型启动子、或者选自PR启动子和PL启动子中的温度诱导型启动子。
发明效果
根据本发明,能够以工业规模高效地生产重组蛋白。
在使用导入有质粒的质粒型表达株的、通过连续培养进行的重组蛋白的表达诱导生产中,利用现有技术时,由于质粒的脱落、结构的不稳定化、表达量的降低等,存在生产率降低的问题。根据本发明,即使对质粒型表达株连续地进行培养,也不会发生质粒的脱落等,还发挥出能够以与染色体中整合有目的蛋白的染色体整合型表达株同等以上的水平稳定、高效地生产重组蛋白的效果。
附图说明
图1是示意性地示出本发明的一个实施方式的重组蛋白的生产方法中使用的培养系统的图。
图2是示出实施例1的反复流加培养所得到的重组改造丝心蛋白的生产量的图。
图3是示出比较例1的反复流加培养所得到的重组改造丝心蛋白的生产量的图。
图4是示意性地示出现有的重组蛋白的生产方法中使用的培养系统的图。
具体实施方式
以下,详细地对本发明的具体实施方式进行说明。但是,本发明并不限定于以下的实施方式。
[重组蛋白的生产方法]
本实施方式的重组蛋白的生产方法是使用表达重组蛋白的重组细胞的生产方法,其特征在于,包括在使重组细胞增殖后的培养液的一部分中添加新鲜的培养基并连续地进行培养的步骤,不重复使用诱导了重组蛋白的表达的重组细胞。本实施方式的生产方法中使用的重组细胞优选为在诱导型启动子的调控下表达重组蛋白的重组细胞。
(重组蛋白)
作为利用本实施方式的生产方法生产的重组蛋白(以下也有时称为“目的蛋白”),可以列举适合以工业规模进行制造的任意的蛋白质,可以列举例如可用于工业用途的蛋白质、可用于医疗用途的蛋白质、结构蛋白等。作为可用于工业用途或医疗用途的蛋白质的具体例,可以列举酶、调控蛋白、受体、肽激素、细胞因子、膜或转运蛋白、用于预防接种的抗原、疫苗、抗原结合蛋白、免疫刺激蛋白、过敏原、全长抗体或抗体片段或者衍生物。作为结构蛋白的具体例,可以列举蛛丝、蚕丝、角蛋白、胶原蛋白、弹性蛋白和节肢弹性蛋白、以及来源于它们的蛋白质等。
作为丝心蛋白样蛋白质的蛛丝或蚕丝来源的蛋白质可以列举例如包含式1:[(A)n基序-REP]m所表示的结构域序列的蛋白质(在此,式1中,(A)n基序表示由4~20个氨基酸残基构成的氨基酸序列,并且(A)n基序中的丙氨酸残基数相对于全部氨基酸残基数为80%以上。REP表示由10~200个氨基酸残基构成的氨基酸序列。m表示8~300的整数。两个以上存在的(A)n基序彼此可以为相同的氨基酸序列,也可以为不同的氨基酸序列。两个以上存在的REP彼此可以为相同的氨基酸序列,也可以为不同的氨基酸序列)。具体而言,可以列举包含序列号1所示的氨基酸序列的蛋白质。
作为胶原蛋白来源的蛋白质,可以列举例如包含式2:[REP2]o所表示的结构域序列的蛋白质(在此,式2中,o表示5~300的整数。REP2表示由Gly-X-Y构成的氨基酸序列,X和Y表示Gly以外的任意的氨基酸残基。两个以上存在的REP2彼此可以为相同的氨基酸序列,也可以为不同的氨基酸序列)。具体而言,可以列举包含序列号2所示的氨基酸序列的蛋白质。序列号2所示的氨基酸序列是在由NCBI数据库获得的人IV型胶原蛋白的部分序列(NCBI的Genbank的登录号:CAA56335.1、GI:3702452)的与重复部分和基序相当的第301位残基至第540位残基的氨基酸序列的N末端附加序列号6所示的氨基酸序列(标签序列和铰链序列)而得到的氨基酸序列。
作为节肢弹性蛋白来源的蛋白质,可以列举例如包含式3:[REP3]p所表示的结构域序列的蛋白质(在此,式3中,p表示4~300的整数。REP3表示由Ser-J-J-Tyr-Gly-U-Pro构成的氨基酸序列。J表示任意的氨基酸残基,特别优选为选自由Asp、Ser和Thr组成的组中的氨基酸残基。U表示任意的氨基酸残基,特别优选为选自由Pro、Ala、Thr和Ser组成的组中的氨基酸残基。两个以上存在的REP3彼此可以为相同的氨基酸序列,也可以为不同的氨基酸序列)。具体而言,可以列举包含序列号3所示的氨基酸序列的蛋白质。序列号3所示的氨基酸序列是在将节肢弹性蛋白(NCBI的Genbank的登录号NP 611157、Gl:24654243)的氨基酸序列中第87位残基的Thr置换为Ser、并且将第95位残基的Asn置换为Asp而得到的序列的第19位残基至第321位残基的氨基酸序列的N末端附加序列号6所示的氨基酸序列(标签序列和铰链序列)而得到的氨基酸序列。
作为弹性蛋白来源的蛋白质,可以列举例如具有NCBI的Genbank的登录号AAC98395(人)、I47076(绵羊)、NP786966(牛)等的氨基酸序列的蛋白质。具体而言,可以列举包含序列号4所示的氨基酸序列的蛋白质。序列号4所示的氨基酸序列是在NCBI的Genbank的登录号AAC98395的氨基酸序列的第121位残基至第390位残基的氨基酸序列的N末端附加序列号6所示的氨基酸序列(标签序列和铰链序列)而得到的氨基酸序列。
作为角蛋白来源的蛋白质,可以列举例如山羊(Capra hircus)的I型角蛋白等。具体而言,可以列举包含序列号5所示的氨基酸序列(NCBI的Genbank的登录号ACY30466的氨基酸序列)的蛋白质。
(表达重组蛋白的重组细胞)
本实施方式的重组细胞例如可以通过利用具有编码目的蛋白的核酸序列和以能够工作的方式与该核酸序列连接的一个或两个以上调节序列的表达载体对宿主进行转化而得到。
调节序列是对宿主中的重组蛋白的表达进行调控的序列(例如启动子、增强子、核糖体结合序列、转录终止序列等),可以根据宿主的种类适当选择。表达载体的种类可以根据宿主的种类适当选择质粒载体、病毒载体、柯斯质粒(cosmid)载体、福斯质粒(fosmid)载体、人工染色体载体等。
作为宿主,原核生物、以及酵母、丝状真菌、昆虫细胞、动物细胞和植物细胞等真核生物均可以优选使用。例如,作为原核生物的优选例,可以列举大肠杆菌、枯草杆菌、假单胞菌、棒杆菌、乳球菌等,更优选可以列举大肠杆菌细胞。
作为表达载体,优选使用在宿主细胞中能够自主复制、或者能够整合到宿主的染色体中、且在能够对编码目的蛋白的核酸进行转录的位置含有诱导型启动子的表达载体。
作为诱导型启动子,只要是在宿主细胞中发挥功能、能够诱导表达目的蛋白的诱导型启动子即可。诱导型启动子是能够根据诱导物质(表达诱导剂)的存在、阻遏分子的非存在、或者温度、渗透压或pH值的升高或降低等物理性因素对转录进行调控的启动子。
作为以原核生物为宿主的情况下的诱导型启动子的具体例,可以列举:由作为乳糖或其类似物的IPTG(异丙基硫代-β-D-半乳糖苷)诱导的T7启动子、tac和trc启动子、lac和lacUV5启动子;由阿拉伯糖诱导的araBAD启动子;由β-吲哚丙烯酸添加或色氨酸饥饿诱导且受色氨酸添加抑制的trp启动子;由鼠李糖诱导的rhaBAD启动子;由木糖诱导的xylF启动子和xylA启动子;由阿拉伯糖诱导的araBAD启动子;由温度升高诱导的λ噬菌体的PR启动子和PL启动子;由磷酸饥饿诱导的phoA启动子;以及由葡萄糖饥饿诱导的cstA启动子和cstA-lacZ启动子等。
作为细菌等原核生物的宿主,可以列举属于埃希氏菌属、短芽孢杆菌属、沙雷氏菌属、芽孢杆菌属、微杆菌属、短杆菌属、棒杆菌属和假单胞菌属等的微生物。
作为属于埃希氏菌属的微生物,可以列举例如大肠杆菌BL21(Novagen公司)、大肠杆菌BL21(DE3)(Life Technologies公司)、大肠杆菌BLR(DE3)(默克密理博公司)、大肠杆菌DH1、大肠杆菌GI698、大肠杆菌HB101、大肠杆菌JM109、大肠杆菌K5(ATCC 23506)、大肠杆菌KY3276、大肠杆菌MC1000、大肠杆菌MG1655(ATCC47076)、大肠杆菌No.49、大肠杆菌Rosetta(DE3)(Novagen公司)、大肠杆菌TB1、大肠杆菌Tuner(Novagen公司)、大肠杆菌Tuner(DE3)(Novagen公司)、大肠杆菌W1485、大肠杆菌W3110(ATCC 27325)、大肠杆菌(Escherichia coli)XL1-Blue、大肠杆菌XL2-Blue等。
作为属于短芽孢杆菌属的微生物,可以列举例如土壤短芽孢杆菌、波茨坦短芽孢杆菌、中孢短芽孢杆菌、美丽短芽孢杆菌、铫子短芽孢杆菌、侧孢短芽孢杆菌、沼泽短芽孢杆菌、副短短芽孢杆菌、罗伊氏短芽孢杆菌、热红短芽孢杆菌、短短芽孢杆菌47(FERM BP-1223)、短短芽孢杆菌47K(FERM BP-2308)、短短芽孢杆菌47-5(FERM BP-1664)、短短芽孢杆菌47-5Q(JCM8975)、桥石短芽孢杆菌HPD31(FERM BP-1087)、桥石短芽孢杆菌HPD31-S(FERMBP-6623)、桥石短芽孢杆菌HPD31-OK(FERM BP-4573)、桥石短芽孢杆菌SP3株(Takara公司制)等。
作为属于沙雷氏菌属的微生物,可以列举例如液化沙雷氏菌(Serratialiquefacience)ATCC14460、嗜虫沙雷氏菌(Serratia entomophila)、无花果沙雷氏菌(Serratia ficaria)、居泉沙雷氏菌(Serratia fonticola)、格氏沙雷氏菌(Serratiagrimesii)、变形斑沙雷氏菌(Serratia proteamaculans)、气味沙雷氏菌(Serratiaodorifera)、普城沙雷氏菌(Serratia plymuthica)、深红沙雷氏菌(Serratia rubidaea)等。
作为属于芽孢杆菌属的微生物,可以列举例如枯草芽孢杆菌(Bacillussubtilis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)等。
作为属于微杆菌属的微生物,可以列举例如嗜氨微杆菌ATCC15354等。
作为属于短杆菌属的微生物,可以列举例如叉开短杆菌(谷氨酸棒杆菌)ATCC14020、黄色短杆菌(谷氨酸棒杆菌ATCC14067)ATCC13826、ATCC14067、乳发酵短杆菌(Brevibacterium immariophilum)ATCC14068、乳酸发酵短杆菌(谷氨酸棒杆菌ATCC13869)ATCC13665、ATCC13869、玫瑰色短杆菌ATCC13825、解糖短杆菌(Brevibacteriumsaccharolyticum)ATCC14066、生硫短杆菌ATCC19240、白色短杆菌ATCC15111、蜡状短杆菌ATCC15112等。
作为属于棒杆菌属的微生物,可以列举例如产氨棒杆菌(Corynebacteriumammoniagenes)ATCC6871、ATCC6872、谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032、谷氨酸棒杆菌ATCC14067、嗜乙酰乙酸棒杆菌(Corynebacteriumacetoacidophilum)ATCC13870、醋谷棒杆菌ATCC15806、解烷棒杆菌ATCC21511、帚石南棒杆菌ATCC15991、谷氨酸棒杆菌ATCC13020,ATCC13032、ATCC13060、百合棒杆菌ATCC15990、栖糖蜜棒杆菌ATCC17965、嗜热产氨棒杆菌AJ12340(FERMBP-1539)、力士棒杆菌ATCC13868等。
作为属于假单胞菌(Pseudomonas)属的微生物,可以列举例如恶臭假单胞菌(Pseudomonas putida)、荧光假单胞菌(Pseudomonas fluorescens)、油菜假单胞菌(Pseudomonas brassicacearum)、黄褐假单胞菌(Pseudomonas fulva)和假单胞菌属菌(Pseudomonas sp.)D-0110等。
作为向上述宿主细胞中导入表达载体的方法,只要是向上述宿主细胞中导入DNA的方法则均可以使用。可以列举例如使用钙离子的方法[Proc.Natl.Acad.Sci.USA,69,2110(1972)]、原生质体法(日本特开昭63-248394)、或Gene,17,107(1982)、Molecular&General Genetics,168,111(1979)中记载的方法等。
属于短芽孢杆菌属的微生物的转化可以利用例如Takahashi等的方法(J.Bacteriol.,1983,156:1130-1134)、Takagi等的方法(Agric.Biol.Chem.,1989,53:3099-3100)、或Okamoto等的方法(Biosci.Biotechnol.Biochem.,1997,61:202-203)来实施。
作为导入编码目的蛋白的核酸的载体(以下简称为“载体”),可以列举例如pBTrp2、pBTac1、pBTac2(均由Boehringer Mannheim公司销售)、pKK233-2(Pharmacia公司制)、pSE280(Invitrogen公司制)、pGEMEX-1(Promega公司制)、pQE-8(QIAGEN公司制)、pKYP10(日本特开昭58-110600号公报)、pKYP200[Agric.Biol.Chem.,48,669(1984)]、pLSA1[Agric.Biol.Chem.,53,277(1989)]、pGEL1[Proc.Natl.Acad.Sci.USA,82,4306(1985)]、pBluescript II SK(-)(Stratagene公司制)、pTrs30[由大肠杆菌JM109/pTrS30(FERM BP-5407)制备]、pTrs32[由大肠杆菌JM109/pTrS32(FERM BP-5408)制备]、pGHA2[由大肠杆菌IGHA2(FERM B-400)制备、日本特开昭60-221091号公报]、pGKA2[由大肠杆菌IGKA2(FERM BP-6798)制备、日本特开昭60-221091号公报]、pTerm2(US4686191、US4939094、US5160735)、pSupex、pUB110、pTP5、pC194、pEG400[J.Bacteriol.,172,2392(1990)]、pGEX(Pharmacia公司制)、pET系统(Novagen公司制)等。
在使用大肠杆菌作为宿主的情况下,可以列举pUC18、pBluescriptII、pSupex、pET22b、pCold等作为优选的载体。
作为属于短芽孢杆菌属的微生物的优选载体的具体例,可以列举作为枯草杆菌载体公知的pUB110、或pHY500(日本特开平2-31682号公报)、pNY700(日本特开平4-278091号公报)、pHY4831(J.Bacteriol.,1987,1239-1245)、pNU200(鹈高重三、日本农艺化学会志1987,61:669-676)、pNU100(Appl.Microbiol.Biotechnol.,1989,30:75-80)、pNU211(J.Biochem.,1992,112:488-491)、pNU211R2L5(日本特开平7-170984号公报)、pNH301(Appl.Environ.Microbiol.,1992,58:525-531)、pNH326、pNH400(J.Bacteriol.,1995,177:745-749)、pHT210(日本特开平6-133782号公报)、pHT110R2L5(Appl.Microbiol.Biotechnol.,1994,42:358-363)、或大肠杆菌与属于短芽孢杆菌属的微生物的穿梭载体pNCO2(日本特开2002-238569号公报)等。
作为真核生物的宿主,可以列举例如酵母和丝状真菌(霉菌等)。
作为酵母,可以列举例如属于酵母(Saccharomyces)属、裂殖酵母(Schizosaccharomyces)属、克鲁维酵母(Kluyveromyces)属、丝孢酵母(Trichosporon)属、许旺酵母(Schwanniomyces)属、毕赤酵母(Pichia)属、假丝酵母(Candida)属、耶氏酵母属和汉逊酵母属等的酵母。更具体而言,可以列举酿酒酵母(Saccharomyces cerevisiae)、粟酒裂殖酵母(Schizosaccharomyces pombe)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxianus)、丛生丝孢酵母(Trichosporon pullulans)、河岸许旺酵母(Schwanniomyces alluvius)、西方许旺酵母(Schwanniomycesoccidentalis)、产朊假丝酵母(Candida utilis)、巴斯德毕赤酵母(Pichia pastoris)、安格斯毕赤酵母(Pichia angusta)、甲醇毕赤酵母(Pichia methanolica)、多形毕赤酵母(Pichia polymorpha)、树干毕赤酵母(Pichia stipitis)、解脂耶氏酵母(Yarrowialipolytica)、多形汉逊酵母(Hansenula polymorpha)等。
使用酵母作为宿主细胞的情况下的表达载体通常优选包含复制起点(需要在宿主中扩增的情况下)和用于大肠杆菌中的载体的增殖的筛选标志物、用于酵母中的重组蛋白表达的诱导型启动子和终止子、以及用于酵母的筛选标志物。
在表达载体为非整合载体的情况下,优选进一步包含自主复制序列(ARS)。由此,能够提高表达载体在细胞内的稳定性(Myers、A.M.、et al.(1986)Gene 45:299-310)。
作为使用酵母作为宿主的情况下的载体,可以列举例如YEP13(ATCC37115)、YEp24(ATCC37051)、YCp50(ATCC37419)、YIp、pHS19、pHS15、pA0804、pHIL3Ol、pHIL-S1、pPIC9K、pPICZα、pGAPZα、pPICZ B等。
作为以酵母为宿主的情况下的诱导型启动子的具体例,可以列举:半乳糖诱导性的gal 1启动子和gal 10启动子;铜诱导性的CUP 1启动子;硫胺诱导性的nmt1启动子;以及甲醇诱导性的AOX1启动子、AOX2启动子、DHAS启动子、DAS启动子、FDH启动子、FMDH启动子、MOX启动子、ZZA1、PEX5-、PEX8-和PEX14-启动子等。
作为向酵母中导入表达载体的方法,只要是将DNA导入酵母中的方法则均可以使用,可以列举例如电穿孔法(Methods Enzymol.,194,182(1990))、原生质球法(Proc.Natl.Acad.Sci.,USA,81,4889(1984))、乙酸锂法(J.Bacteriol.,153,163(1983))、Proc.Natl.Acad.Sci.USA,75,1929(1978)记载的方法等。
作为丝状真菌,可以列举例如属于顶孢霉(Acremonium)属、曲霉(Aspergillus)属、黑粉菌(Ustilago)属、木霉(Trichoderma)属、链孢霉(Neurospora)属、镰孢霉(Fusarium)属、腐质霉(Humicola)属、青霉(Penicillium)属、毁丝霉(Myceliophtora)属、灰霉(Botryts)属、稻瘟霉(Magnaporthe)属、毛霉(Mucor)属、绿僵菌(Metarhizium)属、红曲霉(Monascus)属、根霉(Rhizopus)属和根毛霉属的菌等。
作为丝状真菌的具体例,可以列举阿拉巴马顶孢霉(Acremonium alabamense)、解纤维枝顶孢霉(Acremonium cellulolyticus)、棘孢曲霉(Aspergillus aculeatus)、泡盛曲霉(Aspergillus awamori)、米曲霉(Aspergillus oryzae)、清酒曲霉(Aspergillussake)、酱油曲霉(Aspergillus sojae)、塔宾曲霉(Aspergillus tubigensis)、黑曲霉(Aspergillus niger)、构巢曲霉(Aspergillus nidulans)、寄生曲霉(Aspergillusparasiticus)、无花果曲霉(Aspergillus ficuum)、海枣曲霉(Aspergillus phoeicus)、臭曲霉(Aspergillus foetidus)、黄曲霉(Aspergillus flavus)、烟曲霉(Aspergillusfumigatus)、日本曲霉(Aspergillus japonicus)、绿色木霉(Trichoderma viride)、哈茨木霉(Trichoderma harzianum)、里氏木霉(Trichoderma reseei)、拉克淖金孢子菌(Chrysosporium lucknowense)、嗜热子囊菌(Thermoascus)、孢子丝菌(Sporotrichum)、嗜纤维素侧孢霉(Sporotrichum cellulophilum)、篮状菌(Talaromyces)、太瑞斯梭孢壳霉(Thielavia terrestris)、梭孢壳霉(Thielavia)、粗糙链孢霉(Neurospora crassa)、尖孢镰孢霉(Fusarium oxysporus)、禾谷镰孢霉(Fusarium graminearum)、镶片镰孢霉(Fusarium venenatum)、特异腐质霉(Humicola insolens)、产黄青霉(Penicilliumchrysogenum)、卡门培尔青霉(Penicillium camemberti)、灰白青霉(Penicilliumcanescens)、埃默森青霉(Penicillium emersonii)、绳状青霉(Penicilliumfuniculosum)、灰玫瑰青霉(Penicillium griseoroseum)、产紫青霉(Penicilliumpurpurogenum)、娄地青霉(Penicillium roqueforti)、嗜热毁丝霉(Myceliophtaorathermophilum)、不明毛霉(Mucor ambiguus)、卷枝毛霉(Mucor circinelloides)、易脆毛霉(Mucor fragilis)、冻土毛霉(Mucor hiemalis)、不等孢毛霉(Mucor inaequisporus)、Mucor oblongiellipticus、总状毛霉(Mucor racemosus)、反屈毛霉(Mucor recurvus)、土星状毛霉(Mocor saturninus)、Mocor subtilissmus、多形欧加铁菌(Ogataeapolymorpha)、黄孢原毛平革菌(Phanerochaete chrysosporium)、米黑根毛霉(Rhizomucormiehei)、微小根毛霉(Rhizomucor pusillus)、少根根霉(Rhizopus arrhizus)等。
作为以丝状真菌为宿主的情况下的诱导型启动子的具体例,可以列举:水杨酸诱导性PR1a启动子;环己酰亚胺诱导性Placc启动子;以及奎尼酸诱导性Pqa-2启动子等。
表达载体向丝状真菌中的导入可以使用现有公知的方法进行。可以列举例如Cohen等的方法(氯化钙法)[Proc.Natl.Acad.Sci.USA,69:2110(1972)]、原生质体法[Mol.Gen.Genet.,168:111(1979)]、感受态细胞法[J.Mol.Biol.,56:209(1971)]、电穿孔法等。
本实施方式的重组细胞可以在染色体(染色体DNA)中整合有编码目的蛋白的核酸。编码目的蛋白的核酸以能够工作的方式与一个或两个以上调节序列连接。这种情况下的调节序列可以为外源性的调节序列,也可以为内源性的调节序列。
作为将编码目的蛋白的核酸整合到宿主的染色体中的方法,可以使用公知的方法,可以列举例如应用λ噬菌体的双链切割修复中的重组机制的λred法、Red/ET同源重组法、利用使用pUT-mini Tn5的转座子活性的转移法。另外,例如可以使用BIOMEDAL公司的“利用转座子的基因导入试剂盒:pUTmini-Tn5试剂盒”等,依照试剂盒中记载的方法,将编码目的蛋白的核酸整合到宿主的染色体中。
(培养方法)
本实施方式的生产方法的特征在于,包括在使重组细胞增殖后的培养液的一部分中添加新鲜的培养基并连续地进行培养的步骤,不重复使用诱导了重组蛋白的表达的重组细胞。在此,“不重复使用”是指,在用于再次增殖的培养中不使用诱导了该重组蛋白的表达的重组细胞。培养例如可以在深部通气搅拌培养等需氧条件下进行。
作为本实施方式的培养方法,可以列举例如下述方法:通过分批培养或流加培养使重组细胞增殖后,将包含增殖后的重组细胞的培养液分为重组蛋白的表达诱导用培养液和用于再次增殖的培养用培养液,在用于再次增殖的培养用培养液中添加新鲜的培养基进行培养,并重复上述操作。关于作为重组蛋白的表达诱导用培养液所分取的量,例如,以培养液整体为基准,可以为70~99体积%,优选为80~99体积%,更优选为90~99体积%。关于作为用于再次增殖的培养用培养液所分取的量,例如,以培养液整体为基准,可以为1~30体积%,优选为1~20体积%,更优选为1~10体积%。
在通过流加培养进行用于增殖的培养的情况下,流加基质溶液例如可以包含培养基成分的一种以上的营养素。流加基质溶液的供料按照该技术领域中公知的方法以连续方式、不连续方式等进行即可。对于供料量没有特别限制,将线性常系数方式、线性增加方式、阶梯性增加方式、指数函数的供料方式等组合,以增殖后的菌体量为指标进行供料即可。菌体量例如可以通过干燥菌体重量、湿菌体重量、菌落形成单位等进行确认。通过进行供料,能够高密度地培养重组细胞。
作为其他实施方式的培养方法,可以列举例如下述方法:在一边将培养液量保持大致恒定一边连续地进行新鲜培养基的流加和培养液的排出,在培养液中的培养基组成不随时间变化的稳态下进行培养的培养方法(连续培养)中,将排出的培养液用作重组蛋白的表达诱导用的培养液。
关于培养中使用的培养基的种类没有特别限制。只要是含有重组细胞可同化的碳源、氮源和无机盐类等、能够高效地进行重组细胞的培养的培养基,则天然培养基和合成培养基均可以使用。
作为碳源,只要是重组细胞可同化的碳源即可,可以使用例如葡萄糖、果糖、蔗糖以及含有这些糖的糖蜜、淀粉和淀粉水解物等碳水化合物、乙酸和丙酸等有机酸、以及乙醇和丙醇等醇类。
作为氮源,可以使用例如氨、氯化铵、硫酸铵、乙酸铵和磷酸铵等无机酸或有机酸的铵盐、其他含氮化合物、以及蛋白胨、肉提取物、酵母提取物、玉米浆、酪蛋白水解物、豆粕和豆粕水解物、各种发酵菌体及其消化产物。
作为无机盐,可以使用例如磷酸二氢钾、磷酸氢二钾、磷酸镁、硫酸镁、氯化钠、硫酸亚铁、硫酸锰、硫酸铜和碳酸钙。
培养温度例如为15~40℃。培养中的培养液的pH优选保持于3.0~9.0。培养液的pH可以使用无机酸、有机酸、碱溶液、尿素、碳酸钙和氨等进行调节。
参考图1对一个实施方式的生产方法更具体地进行说明。图1是示意性地示出本发明的一个实施方式的重组蛋白的生产方法中使用的培养系统的图。图1所示的培养系统100具备培养槽10和培养槽20这两个培养槽。培养槽10是使重组细胞增殖的培养槽。从经由泵50与培养槽10连接的流加基质溶液储存槽30向培养槽10中供给流加基质溶液31。
在培养槽10中增殖后的重组细胞例如在从对数生长期的中期进入后期的时刻作为培养液11被转移至培养槽20中。关于所转移的培养液11的量,例如以培养液11的总量为基准,可以为70~99体积%,优选为80~99体积%,更优选为90~99体积%。在转移后的培养槽10中残留有一部分培养液11,从培养基储存槽40向其中供给新鲜的培养基41,重复进行重组细胞的增殖。
培养槽20是使重组蛋白表达的培养槽。在培养槽20中,使诱导型启动子活化,诱导重组蛋白的表达。在此,诱导型启动子的活化是指,使利用诱导型启动子的编码重组蛋白的核酸的转录活化。例如在诱导型启动子为在诱导物质(表达诱导剂)存在下活化的启动子的情况下,可以通过向培养槽20中添加该诱导物质而诱导重组蛋白的表达。另外,例如在诱导型启动子为通过温度的升高或降低而活化的启动子的情况下,可以通过将培养槽20加温或冷却、对培养液21的温度进行控制来诱导重组蛋白的表达。
在培养槽20中,例如在10~20小时的期间诱导重组蛋白的表达。在此期间,可以从经由泵51连接的流加基质溶液储存槽30向培养槽20中供给流加基质溶液31。流加基质溶液31包含培养用培养基中含有一种以上的营养素。通过供给流加基质溶液31,重组蛋白的表达诱导的效率提高。
在进行期望时间的重组蛋白的表达诱导后,培养液21被转移至分离纯化槽60中。关于所转移的培养液21的量,以培养液21的总量为基准,例如可以为50~100体积%,优选为80~100体积%,更优选为90~100体积%,进一步优选为100体积%。在分离纯化槽60中,对表达后的重组蛋白进行分离和纯化。
向培养液21被转移后的培养槽20中转移在培养槽10中增殖后的重组细胞,诱导重组蛋白的表达。这样,连续地重复进行在培养槽10中的重组细胞的增殖和在培养槽20中的重组蛋白的表达诱导。
培养系统100中,由于重组细胞的增殖与重组蛋白的表达诱导在不同的培养槽(培养槽10和培养槽20)中进行,因此诱导了重组蛋白的表达的重组细胞不会被重复使用。根据该构成,即使重复进行上述循环,也能够稳定且高效地生产重组蛋白。另外,在使用质粒型表达株作为重组细胞的情况下,还抑制质粒的脱落等。
图4是示意性地示出现有的重组蛋白的生产方法中使用的培养系统的图。图4所示的培养系统200仅具备一个培养槽10。在培养槽10中,同时进行重组细胞的增殖和重组蛋白的表达诱导。在培养槽10中进行期望时间的重组蛋白的表达诱导后,培养液11被转移至分离纯化槽60中。转移后的培养槽10中残留有一部分培养液11,从培养基储存槽40向其中供给新鲜的培养基41,重复进行重组细胞的增殖和重组蛋白的表达诱导。培养系统200的构成中,在重复进行上述循环的情况下重组蛋白的生产量显著降低。另外,在使用质粒型表达株作为重组细胞的情况下,容易发生质粒的脱落。
一个实施方式的生产方法可以包括:重复进行下述(A)~(E)步骤。
(A)通过分批培养或流加培养使重组细胞在培养槽中增殖的步骤
(B)在(A)步骤中的增殖后,将培养槽的培养液的一部分转移至接收用培养槽中的步骤
(C)在(B)步骤中的转移后,在两个培养槽中的任意一个中添加新鲜的培养基,进入(A)步骤的步骤
(D)在(C)步骤中未进入(A)步骤的另一个培养槽中诱导上述重组蛋白的表达,使上述重组蛋白蓄积的步骤
(E)从培养液中分离和纯化(D)步骤中蓄积的上述重组蛋白的步骤
在接收用培养槽中实施(D)步骤的情况下,关于在(B)步骤中转移至接收用培养槽中的培养液的量,以培养液总量为基准,可以为70~99体积%,优选为80~99体积%,更优选为90~99体积%。另外,在接收用培养槽中实施(C)步骤的情况下,关于在(B)步骤中转移至接收用培养槽中的培养液的量,以培养液总量为基准,可以为1~30体积%,优选为1~20体积%,更优选为1~10体积%。
(重组蛋白的表达诱导)
重组蛋白的表达的诱导通过使利用诱导型启动子的转录(编码目的蛋白的核酸的转录)活化而进行。诱导型启动子的活化可以根据诱导型启动子的种类按照该技术领域中公知的方法进行。
例如,在使用通过诱导物质(表达诱导剂)的存在而活化的诱导型启动子的情况下,可以通过在培养液中添加该诱导物质而诱导重组蛋白的表达。诱导物质可以一次性或分成多次添加至培养液中,另外也可以通过连续供料而添加至培养液中。也可以使流加基质溶液中含有诱导物质而进行供料。添加的诱导物质的量可以根据诱导物质和诱导型启动子的种类来设定,例如相对于重组细胞的每1g干燥重量可以设定为0.1~30μg的范围,优选为0.5~20μg的范围。
另外,例如在使用通过温度的升高或降低而活化的诱导型启动子的情况下,可以通过使培养液的温度升高或降低而诱导重组蛋白的表达。例如在使用通过温度升高而活化的λ噬菌体的PR启动子或PL启动子的情况下,可以通过使增殖时的培养液的温度为20~37℃的范围而抑制增殖时的重组蛋白的表达,接着通过使培养液的温度升高至38~44℃而诱导重组蛋白的表达。此时,为了缓和由热休克蛋白产生的影响,如日本特开平6-292563号公报所记载的那样使增殖时的培养液的pH为6.5~7.5,在开始重组蛋白的表达诱导的时刻使培养液的pH变动为4.5~6.5,由此,能够进行更稳定的表达诱导。
从进行重组细胞的增殖的阶段向诱导重组蛋白的表达的阶段移行的时期没有特别限制,可以根据培养系统的构成、生产工艺的设计进行适当设定。从高效地进行重组蛋白的生产的观点出发,优选在重组细胞的增殖达到对数生长期的中期至后期时开始重组蛋白的表达的诱导。
重组细胞的增殖从延滞期或诱导期(培养初期的细胞数的增加慢的时期)开始,经过对数生长期(每单位时间细胞数增加2倍地以对数增加的时期),达到稳定期(细胞的净数量观察不到变动的时期)。对数生长期的中期是指达到延滞期的细胞数与稳定期的细胞数的中间程度的细胞数的时期,对数生长期的后期是指从中期到稳定期为止的时期。作为开始重组蛋白的表达的诱导的时期的具体例,例如在稳定期中的OD 600的值为约150的重组细胞的情况下,优选为OD 600的值达到30~110的时期,更优选为OD 600的值达到40~90的时期,进一步优选为OD 600的值达到50~80的时期。
诱导重组蛋白的表达的时间根据所使用的宿主、目的蛋白的种类进行至达到设定的生产量为止即可。由于生产速度根据培养液的温度等培养条件而变化,因此不需要一概地决定诱导重组蛋白的表达的时间。可以配合下一步骤的重组蛋白的分离和纯化的进行来设定诱导重组蛋白的表达的时间。另外,在工业生产上优选按照不会对并行进行的重组细胞的增殖和该增殖后的重组细胞的转移产生影响的方式设定诱导重组蛋白的表达的时间。
进行了重组蛋白的表达诱导的培养液用于下述目的重组蛋白的分离、纯化。
(重组蛋白的分离、纯化)
重组蛋白的分离和纯化可以利用通常使用的方法进行。例如,在重组蛋白以溶解状态在细胞内进行表达的情况下,在用于重组蛋白的表达诱导的培养结束后,通过离心分离回收宿主细胞(重组细胞),悬浮于水系缓冲液中后,利用超声波破碎机、弗氏压碎器、Manton-Gaulin匀浆器和戴诺研磨机(DYNO-MILL)等将宿主细胞破碎,得到无细胞提取液。从通过将该无细胞提取液离心分离而得到的上清中,单独或组合使用蛋白质的分离纯化中通常使用的方法、即溶剂提取法、利用硫酸铵等的盐析法、脱盐法、利用有机溶剂的沉淀法、使用二乙基氨基乙基(DEAE)-琼脂糖、DIAION HPA-75(三菱化成公司制)等树脂的阴离子交换层析法、使用S-琼脂糖FF(Pharmacia公司制)等树脂的阳离子交换层析法、使用丁基琼脂糖、苯基琼脂糖等树脂的疏水性层析法、使用分子筛的凝胶过滤法、亲和层析法、色谱聚焦法、等电点电泳等电泳法等方法,能够得到重组蛋白的纯化制备品。
另外,在重组蛋白在细胞内形成不溶体而进行表达的情况下,同样在回收宿主细胞后将其破碎并进行离心分离,由此以沉淀级分的形式回收重组蛋白的不溶体。回收的重组蛋白的不溶体可以利用蛋白变性剂进行可溶化。该操作后,利用与上述同样的分离纯化法能够得到重组蛋白的纯化制备品。
在重组蛋白被分泌到细胞外的情况下,可以从培养上清中回收重组蛋白。即,利用离心分离等方法对培养液进行处理,由此获得培养上清,通过使用与上述同样的分离纯化法,能够从该培养上清中得到重组蛋白的纯化制备品。
实施例
以下,基于实施例等更具体地对本发明进行说明。但是,本发明并不限定于以下的实施例。
[实施例1]
[(1)改造丝心蛋白表达株的制作]
(质粒型表达株的制作)
基于金纺蜘蛛(Nephila clavipes)来源的丝心蛋白(GenBank登录号:P46804.1、GI:1174415)的碱基序列和氨基酸序列,设计出具有序列号1所示的氨基酸序列的改造丝心蛋白(以下也称为“SEC472”)。
序列号1所示的氨基酸序列具有以生产率的提高为目的而对金纺蜘蛛来源的丝心蛋白的氨基酸序列实施了氨基酸残基的置换、插入和缺失的氨基酸序列,并进一步在N末端附加有序列号6所示的氨基酸序列(标签序列和铰链序列)。
接着,合成编码SEC472的核酸。该核酸中,在5’末端附加有NdeI位点、在终止密码子下游附加有EcoRI位点。将该核酸克隆至克隆载体(pUC118)中。然后,利用NdeI和EcoRI对该核酸进行限制酶处理并将其切出后,重组到蛋白表达载体pET-22b(+)中,得到表达载体。
利用pET-22b(+)表达载体对大肠杆菌BLR(DE3)进行转化,获得导入有质粒的质粒型表达株SEC659。
(染色体整合型表达株的制作)
使用BIOMEDAL公司的pUTmini-Tn5试剂盒,制作染色体整合型表达株。
合成编码具有序列号1所示的氨基酸序列的改造丝心蛋白(SEC472)的核酸。该核酸中,在5’末端和终止密码子下游附加有NotI位点。制作将该核酸插入到pUTmini-Tn5Km的NotI位点而得到的质粒。接着,将利用该质粒转化S17-1λpir后得到的株与大肠杆菌BLR(DE3)以1:1混合,在含有LB和Km的平板培养基上培养。从显示出Km耐性和Ap敏感性的株中获得在染色体中整合有该核酸的染色体整合型表达株SEC714。
[(2)种子培养]
将上述(1)中制作的质粒型表达株SEC659和染色体整合型表达株SEC714分别在包含氨苄青霉素的2mL的LB培养基中培养15小时。将该培养液添加到包含氨苄青霉素的100mL的种子培养用培养基(表1)中,使OD 600为0.005,将培养液温度保持于30℃,进行烧瓶培养直至OD 600达到5(约15小时)为止,得到各自的种子培养液。
[表1]
种子培养用培养基
[(3)主培养用培养基的制备]
将主培养用培养基的组成示于表2。
[表2]
主培养用培养基
向培养槽中添加500mL的主培养用培养基(表2),利用高压釜(TOMY LSX-500)在121℃下进行20分钟灭菌处理。冷却至37℃后,使用28-30%氨水(关东化学、01266-88)将pH调节至6.1~6.3。
[(4)流加基质溶液的制备]
将流加基质溶液的组成示于表3。
[表3]
流加基质溶液
向流加罐中添加规定量的流加基质溶液,利用高压釜(TOMY LSX-500)在121℃下进行20分钟灭菌处理。
[(5)反复流加培养和表达诱导]
使用图1所示的培养系统100,利用质粒型表达株SEC659和染色体整合型表达株SEC714进行重组改造丝心蛋白的生产。作为培养槽10和培养槽20,使用TSC-A1L-5型培养槽(高杉制作所、容量1L)。重组改造丝心蛋白通过反复流加培养进行生产,反复流加培养中,将流加培养至达到规定细胞密度的培养液(约95%)用于表达诱导,在剩余的培养液(约5%)中添加新鲜的培养基并反复进行流加培养。
将主培养用培养基(初始培养基量0.5L)倒入培养槽10中,添加上述得到的种子培养液,使OD 600为0.05。将培养液的温度保持于37℃,使用30%氨水和4M磷酸溶液(和光纯药工业)在pH6.9下控制恒定而进行主培养。另外,进行通气搅拌,以将培养液中的溶解氧浓度维持于溶解氧饱和浓度的30~40%。这些控制使用质量流量控制器(阿自倍尔、MPC0005BBRN0100D0)。
在主培养中,在低于溶解氧饱和浓度的约30%后,在超过溶解氧饱和浓度的约55%的时刻从流加基质溶液储存槽30向培养槽10开始进行流加基质溶液31的流加。流加基质溶液31的流加速度设定为6g/小时的恒速流加。
在培养槽10中,继续流加培养至培养液11的OD 600达到约60为止,然后,将该培养液11的约95%经由进样管线(未图示)无菌地转移至培养槽20中。转移后,在培养槽20中以达到0.2mM的方式添加IPTG(表达诱导剂),开始重组改造丝心蛋白的表达诱导。
表达诱导开始后,以9g/小时的流加速度从流加基质溶液储存槽30向培养槽20进行流加基质溶液31的流加,除此以外,在与培养槽10中的主培养相同的条件下继续培养。在培养槽20中进行约16小时表达诱导后,将全部的培养液21转移至分离纯化槽60中,用于重组改造丝心蛋白的分离、纯化。
从培养基储存槽40向将培养液11转移至培养槽20中后的培养槽10(残留有约5%的培养液11)中添加新鲜的培养基41,在与上述同样的条件下在培养槽10中进行主培养和流加培养。向将全部的培养液21转移至分离纯化槽60中后的培养槽20中转移与上述同样地流加培养至OD600达到约60为止的培养槽10的培养液11,进行重组改造丝心蛋白的表达诱导。包括上述主培养在内,重复进行8次该反复流加培养。
对利用质粒型表达株SEC659和染色体整合型表达株SEC714进行的8次反复流加培养中的重组改造丝心蛋白的生产量进行分析,将结果示于图2。图2所示的生产量以将利用SEC714进行的第1次反复流加培养中的重组改造丝心蛋白的生产量设为100%时的相对值表示。
如图2所示,质粒型表达株SEC659和染色体整合型表达株SEC714这两株均在第8次反复流加培养中也维持了第1次反复流加培养中的重组改造丝心蛋白的生产量。
另外,利用下述方法对质粒型表达株SEC659中的质粒保持率进行确认。将反复流加培养的各次培养后的培养液用LB培养基稀释,接种到LB琼脂培养基(培养基A)上。在37℃的恒温槽中培养18~20小时后,将生长出的50~100个菌落用灭菌完毕的牙签转移至添加有氨苄青霉素的LB琼脂培养基(培养基B)上。将移植至该培养基B上的菌株在37℃的恒温槽中培养12~18小时后,计数从培养基A转移至培养基B的菌落数和在培养基B上形成的菌落数,利用下述计算式算出质粒保持率。
质粒保持率=(在培养基B上形成的菌落数)/(从培养基A转移至培养基B的菌落数)
其结果是,质粒型表达株SEC659直至第8次反复流加培养的IPTG添加时为止维持了100%的质粒保持率。
需要说明的是,对于包含序列号2~5中任一者所示的氨基酸序列的、角蛋白、胶原蛋白、弹性蛋白和节肢弹性蛋白来源的重组蛋白,利用上述方法也能够持续进行稳定的生产。
[比较例1]
[反复流加培养和表达诱导]
使用图4所示的现有的培养系统200,利用质粒型表达株SEC659和染色体整合型表达株SEC714进行重组改造丝心蛋白的生产。作为培养槽10和培养槽20,使用TSC-A1L-5型培养槽(高杉制作所、容量1L)。重组改造丝心蛋白通过反复流加培养进行生产,该反复流加培养中,在进行了表达诱导的培养液的一部分(约5%)中添加新鲜的培养基并反复进行流加培养。
不在培养槽20中、而是在进行了主培养和流加培养的培养槽10中继续进行重组改造丝心蛋白的表达诱导,除此以外,在与实施例1基本相同的条件下进行反复流加培养和表达诱导。
即,在培养槽10中继续流加培养至培养液11的OD 600达到约60为止,然后,接着以达到0.2mM的方式在培养槽10中添加IPTG(表达诱导剂),开始重组改造丝心蛋白的表达诱导。表达诱导开始后,以9g/小时的流加速度从流加基质溶液储存槽30向培养槽10进行流加基质溶液31的流加。在培养槽10中进行约16小时表达诱导后,将培养液11的约95%转移至分离纯化槽60中,用于重组改造丝心蛋白的分离、纯化。转移后,从培养基储存槽40向该培养槽10(残留有约5%的培养液11)中添加新鲜的培养基41,在相同的条件下进行主培养、流加培养和重组改造丝心蛋白的表达诱导。重复5次该反复流加培养。
对利用质粒型表达株SEC659和染色体整合型表达株SEC714进行的5次反复流加培养中的重组改造丝心蛋白的生产量进行分析,将结果示于图3。图3所示的生产量以将利用SEC714进行的第1次反复流加培养中的重组改造丝心蛋白的生产量设为100%时的相对值表示。
如图3所示,在仅在培养槽10中重复进行反复流加培养的情况下(即,重复使用诱导了重组改造丝心蛋白的表达的重组细胞的情况下),在质粒型表达株SEC659中,第2次以后的重组改造丝心蛋白的生产量极度降低。与实施例1同样测定的质粒保持率在第2次反复流加培养的IPTG添加时刻变为0%。另一方面,在染色体整合型表达株SEC714中,重组改造丝心蛋白的生产量虽然随着反复流加培养的次数增加而降低,但与质粒型表达株SEC659相比显示出稳定的生产量。
标号说明
10、20…培养槽、11、21…培养液、30…流加基质溶液储存槽、31…流加基质溶液、40…培养基储存槽、41…培养基、50、51…泵、60…分离纯化槽、100、200…培养系统。
序列表
<110> 丝芭博株式会社(Spiber Inc.)
小岛冲压工业株式会社(KOJIMA INDUSTRIES CORPORATION)
<120> 重组蛋白的生产方法(A method of producing recombinant protein)
<130> FP17-0434-00
<160> 6
<170> PatentIn version 3.5
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Glu Asn Glu Leu Ala Leu His Gln Ser Val Glu Ala Asp Ile Asn Gly
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Leu His Arg Val Met Asp Glu Leu Thr Leu Cys Thr Ser Asp Leu Glu
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Claims (11)
1.一种重组蛋白的生产方法,其为使用在诱导型启动子的调控下表达重组蛋白的重组细胞的该重组蛋白的生产方法,其中,
包括在使所述重组细胞增殖后的培养液的一部分中添加新鲜的培养基并连续地进行培养的步骤,
不重复使用诱导了所述重组蛋白的表达的重组细胞。
2.如权利要求1所述的重组蛋白的生产方法,其中,所述重组细胞的增殖和所述重组蛋白的表达的诱导在不同的培养槽中进行。
3.如权利要求1或2所述的重组蛋白的生产方法,其中,所述重组细胞的增殖通过流加培养进行。
4.一种重组蛋白的生产方法,其为使用在诱导型启动子的调控下表达重组蛋白的重组细胞的该重组蛋白的生产方法,其中,
包括重复进行下述(A)~(E)步骤:
(A)通过分批培养或流加培养使所述重组细胞在培养槽中增殖的步骤;
(B)在(A)步骤中的增殖后,将所述培养槽的培养液的一部分转移至接收用培养槽中的步骤;
(C)在(B)步骤中的转移后,在两个培养槽中的任意一个中添加新鲜的培养基,进入(A)步骤的步骤;
(D)在(C)步骤中未进入(A)步骤的另一个培养槽中诱导所述重组蛋白的表达,使所述重组蛋白蓄积的步骤;
(E)从培养液中分离和纯化(D)步骤中蓄积的所述重组蛋白的步骤。
5.如权利要求1~4中任一项所述的重组蛋白的生产方法,其中,在所述重组细胞的增殖达到对数生长期的中期时,开始所述重组蛋白的表达的诱导。
6.如权利要求1~5中任一项所述的重组蛋白的生产方法,其中,所述重组蛋白的表达的诱导通过在培养液中添加表达诱导剂、或者改变培养液的温度来进行。
7.如权利要求4所述的重组蛋白的生产方法,其中,以培养液总量为基准,在(B)步骤中转移至接收用培养槽中的培养液的量为80~99体积%。
8.如权利要求1~7中任一项所述的重组蛋白的生产方法,其中,所述重组细胞为编码所述重组蛋白的基因被整合至染色体中的细胞。
9.如权利要求1~8中任一项所述的重组蛋白的生产方法,其中,所述重组蛋白为结构蛋白。
10.如权利要求9所述的重组蛋白的生产方法,其中,所述结构蛋白为来源于选自由角蛋白、胶原蛋白、弹性蛋白、节肢弹性蛋白、蚕丝和蛛丝组成的组中的蛋白质的蛋白质。
11.如权利要求1~10中任一项所述的重组蛋白的生产方法,其中,所述诱导型启动子为选自T7启动子、tac启动子、trc启动子、lac启动子和lacUV5启动子中的IPTG诱导型启动子、或者选自PR启动子和PL启动子中的温度诱导型启动子。
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CN110938614A (zh) * | 2019-12-06 | 2020-03-31 | 宁波希诺亚海洋生物科技有限公司 | 高活性β-半乳糖苷酶、高通量筛选该酶的质粒及其制备方法 |
CN118480554A (zh) * | 2024-07-16 | 2024-08-13 | 苏州拾光医药生物科技有限公司 | 重组弹性蛋白及其表达体系和应用 |
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WO2020158874A1 (ja) * | 2019-01-31 | 2020-08-06 | Spiber株式会社 | 組換えタンパク質の製造方法 |
CN113439087A (zh) * | 2019-01-31 | 2021-09-24 | 丝芭博株式会社 | 重组蛋白的制备方法 |
WO2021020546A1 (ja) * | 2019-08-01 | 2021-02-04 | Spiber株式会社 | 組換え細胞及び組換えタンパク質の製造方法 |
WO2023180525A1 (en) * | 2022-03-24 | 2023-09-28 | Richter Gedeon Nyrt. | Method for the manufacture of biopharmaceuticals |
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