CN114686548A - 用于增强生产的rna相关酶的修饰 - Google Patents
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Abstract
本发明尤其提供了使用SUMO‑鸟苷酰转移酶融合蛋白用于大规模生产加帽的mRNA的方法和组合物。
Description
本申请是申请号为201680060626.9、申请日为2016年10月14日、发明名称为“用于增强生产的RNA相关酶的修饰”的中国发明专利申请的分案申请,原申请为国际申请号为PCT/US2016/057044的国家阶段申请,该国际申请要求2015年10月14日提交的美国临时专利申请第62/241,350号的优先权。
相关专利申请
本专利申请要求于2015年10月14日提交的美国临时申请序列号62/241,350的优先权,所述美国临时申请的公开内容在此以引用的方式并入。
序列表
本说明书参考了于2016年10月14日作为命名为“SL_SHR-1187WO”的.txt文件电子提交的序列表。该.txt文件于2016年10月14日生成,并且大小为28,402字节。序列表的全部内容以引用的方式并入本文。
背景技术
信使RNA(“mRNA”)疗法正在成为用于治疗各种疾病的越来越重要的方法。有效的mRNA疗法需要将mRNA有效递送给患者,以及由mRN A编码的蛋白质在患者体内的有效产生。为了最佳化mRNA递送和体内蛋白质产生,在构建体的5'末端处通常需要适当的帽,所述帽保护mRNA免于降解并且促进成功的蛋白质翻译。因此,能够给mRNA加帽的酶的大规模生产对于产生用于治疗应用的mRNA特别重要。
发明内容
本发明提供了用于有效生产能够给mRNA加帽的酶的改进方法。本发明部分基于下述令人惊讶的发现:用SUMO标签修饰鸟苷酰转移酶(GT)使得以产生用于治疗应用的加帽的mRNA所需的大规模生产GT成为可能。
因此,在一个方面,本发明提供了产生加帽的RNA或RNA类似物寡核苷酸的方法,其中融合蛋白促进将鸟苷酰分子转移至RNA或RNA类似物寡核苷酸的5’末端和使鸟苷酰分子甲基化的步骤。
在一些实施例中,融合蛋白包含鸟苷酰转移酶和小泛素样分子(SUMO)蛋白。在一些实施例中,鸟苷酰转移酶包含SEQ ID NO:6和SEQ ID NO:7,并且SUMO蛋白包含SEQ IDNO:1。在一些实施例中,融合蛋白包含SEQ ID NO:8和SEQ ID NO:7。
在一些实施例中,RNA或RNA类似物寡核苷酸的一个末端是5'末端。
在一些实施例中,相对于野生型鸟苷酰转移酶蛋白,融合蛋白具有可比较的磷酸酶活性、鸟苷酰转移酶活性和甲基化活性。
在另一个方面,本发明提供了融合蛋白,其中融合蛋白包含鸟苷酰转移酶和小泛素样分子(SUMO)蛋白。
在一些实施例中,鸟苷酰转移酶包含SEQ ID NO:6和SEQ ID NO:7,并且SUMO蛋白包含SEQ ID NO:1。在一些实施例中,鸟苷酰转移酶包含大亚基和小亚基。在一些实施例中,SUMO蛋白与大亚基共价连接且共表达。在一些实施例中,相对于野生型鸟苷酰转移酶蛋白,融合蛋白具有可比较的磷酸酶活性、鸟苷酰转移酶活性和甲基化活性。
在另一个方面,本发明提供了编码包含鸟苷酰转移酶蛋白和小泛素样分子(SUMO)蛋白的融合蛋白的载体。
在一些实施例中,载体包含SEQ ID NO:5和SEQ ID NO:2。在一些实施例中,载体包含SEQ ID NO:5、SEQ ID NO:2和SEQ ID NO:3。在一些实施例中,载体包含SEQ ID NO:4和SEQ ID NO:3。
在另一个方面,本发明提供了通过发酵生产鸟苷酰转移酶的方法,所述方法包括:a)在发酵培养基中培养用至少一种重组核酸分子转化的微生物,所述重组核酸分子包含编码鸟苷酰转移酶的核酸序列,所述鸟苷酰转移酶具有与SEQ ID NO:6和SEQ ID NO:7至少90%相同的氨基酸序列;和b)收集由培养步骤产生的产物。
在一些实施例中,鸟苷酰转移酶包含鸟苷酰转移酶融合蛋白。在一些实施例中,相对于野生型鸟苷酰转移酶蛋白,鸟苷酰转移酶融合蛋白具有可比较的磷酸酶活性、鸟苷酰转移酶活性和甲基化活性。在一些实施例中,鸟苷酰转移酶融合蛋白包含小泛素样分子(SUMO)蛋白。在一些实施例中,鸟苷酰转移酶融合蛋白包含SEQ ID NO:8。
在一些实施例中,SUMO蛋白通过共价键与鸟苷酰转移酶结合。在一些实施例中,共价键在SUMO蛋白和鸟苷酰转移酶的大亚基之间。
在一些实施例中,发酵培养基选自Terrific Broth、Cinnabar、2xYT和LB。在一些实施例中,微生物是细菌。
在一些实施例中,编码鸟苷酰转移酶的核酸序列与SEQ ID NO:2和SEQ ID NO:3至少90%相同。
在一些实施例中,重组核酸分子还包含编码小泛素样分子(SUMO)蛋白的核酸序列。在一些实施例中,编码小泛素样分子(SUMO)蛋白的核酸序列与SEQ ID NO:5至少90%相同。
在一些实施例中,产物是鸟苷酰转移酶。在一些实施例中,产物是包含鸟苷酰转移酶融合蛋白的鸟苷酰转移酶。在一些实施例中,鸟苷酰转移酶融合蛋白还包含小泛素样分子(SUMO)蛋白。
附图说明
这些附图用于说明的目的并且决不是限制性的。
图1A和1B是本发明的各种实施例中存在的示例性mRNA加帽结构的图解。
图2证实了与经由摇瓶方法产生的GT蛋白的产率相比,通过发酵产生的可溶性SUMO-GT蛋白的示例性产率。
定义
为了使本发明更容易理解,首先定义了某些术语。下述术语和其他术语的另外定义在说明书自始至终得到阐述。
大约:如本文使用的,当应用于一个或多个目的值时,术语“大约”或“约”指与所述参考值类似的值。在某些实施例中,术语“大约”或“约”指在所述参考值的任一方向上(大于或小于)落入25%、20%、19%、18%、17%、16%、15%、14%、13%、12%、11%、10%、9%、8%、7%、6%、5%、4%、3%、2%、1%或更少内的一系列值,除非另有说明或从上下文另外显而易见(除非当这种数目超过可能值的100%时)。
分批培养:如本文使用的,术语“分批培养”指培养细胞的方法,其中最终用于培养细胞的所有组分,包括培养基(参见下文“培养基”的定义)以及细胞本身,在培养过程开始时提供。因此,分批培养通常指允许从接种进行到结束的培养,而无需用新鲜培养基给培养的细胞重新补料。分批培养通常在某个点停止,并且收获培养基中的细胞和/或组分且任选进行纯化。
生物活性的:如本文使用的,短语“生物活性的”指在生物系统(例如细胞培养物、生物等)中具有活性的任何物质的特征。例如,当被施用于生物时,对该生物具有生物作用的物质被视为生物活性的。生物活性还可通过体外测定(例如体外酶促测定,如硫酸盐释放测定)来测定。在特定实施例中,当蛋白质或多肽是生物活性的时,共享蛋白质或多肽的至少一种生物活性的该蛋白质或多肽的一部分通常称为“生物活性”部分。在一些实施例中,蛋白质由细胞培养系统产生和/或纯化,所述细胞培养系统当施用于受试者时展示生物活性。在一些实施例中,蛋白质需要进一步加工以便成为生物活性的。在一些实施例中,蛋白质需要翻译后修饰,例如但不限于糖基化(例如唾液酸化)、法尼基化、切割、折叠、甲酰甘氨酸转换及其组合,以便成为生物活性的。在一些实施例中,作为前体形式(proform)(即未成熟形式)产生的蛋白质可能需要另外的修饰,以成为生物活性的。
生物反应器:如本文使用的,术语“生物反应器”指用于宿主细胞培养物生长的容器。生物反应器可具有任何尺寸,只要它可用于培养哺乳动物细胞。通常,生物反应器为至少1升,并且可为10、100、250、500、1000、2500、5000、8000、10,000、12,0000升或更多,或者之间的任何体积。生物反应器的内部条件,包括但不限于pH、摩尔渗透压浓度、CO2饱和度、O2饱和度、温度及其组合,通常在培养期间加以控制。生物反应器可由任何材料组成,所述材料适合于在本发明的培养条件下将细胞保持在培养基中,包括玻璃、塑料或金属。在一些实施例中,生物反应器可用于执行动物细胞培养。在一些实施例中,生物反应器可用于执行哺乳动物细胞培养。在一些实施例中,生物反应器可与源自生物的细胞和/或细胞系一起使用,所述细胞例如但不限于哺乳动物细胞、昆虫细胞、细菌细胞、酵母细胞和人细胞。在一些实施例中,生物反应器用于大规模细胞培养生产,并且通常为至少100升,并且可为200、500、1000、2500、5000、8000、10,000、12,0000升或更多,或者之间的任何体积。本领域普通技术人员将意识到并且能够选择用于实践本发明的合适的生物反应器。
细胞密度:如本文使用的,术语“细胞密度”指在给定体积的培养基中存在的细胞数目。
细胞培养或培养:如本文使用的,这些术语指在适合于细胞群体存活和/或生长的条件下,在培养基中生长的细胞群体。如本领域普通技术人员将清楚的,如本文使用的,这些术语可指包含细胞群体和群体在其中生长的培养基的组合。
培养:如本文使用的,术语“培养”或语法等价物指在有利于生长或存活的条件下维持细胞的过程。术语“培养”和“细胞培养”或任何同义词在本专利申请中可互换使用。
培养容器:如本文使用的,术语“培养容器”指可提供用于培养细胞的无菌环境的任何容器。示例性培养容器包括但不限于玻璃、塑料或金属容器。
表达:如本文使用的,核酸序列的“表达”指下述事件中的一个或多个:(1)从DNA序列产生RNA模板(例如通过转录);(2)RNA转录物的加工(例如通过剪接、编辑、5'帽形成和/或3’末端形成);(3)RNA翻译成多肽或蛋白质;和/或(4)多肽或蛋白质的翻译后修饰。
补料分批培养:如本文使用的,术语“补料分批培养”指培养细胞的方法,其中在培养过程开始之后的某个时间将另外的组分提供给培养物。所提供的组分通常包含在培养过程期间已耗尽的用于细胞的营养补充剂。补料分批培养通常在某个点停止,并且收获培养基中的细胞和/或组分并且任选进行纯化。
同源性:如本文使用的,术语“同源性”指聚合物分子之间,例如核酸分子(例如DNA分子和/或RNA分子)之间和/或多肽分子之间的总体相关性。在一些实施例中,如果聚合物分子的序列至少25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或99%相同,则聚合物分子被视为彼此“同源”。在一些实施例中,如果聚合物分子的序列至少25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或99%相似,则聚合物分子被视为彼此“同源”。
同一性:如本文使用的,术语“同一性”指聚合物分子之间,例如核酸分子(例如DNA分子和/或RNA分子)之间和/或多肽分子之间的总体相关性。两个核酸序列的同一性百分比的计算例如可通过就最佳比较目的比对两个序列来执行(例如可将缺口引入第一核酸序列和第二核酸序列之一或两者中用于最佳比对,并且出于比较目的可忽略不相同的序列)。在某些实施例中,为了比较目的比对的序列的长度为参考序列的长度的至少30%、至少40%、至少50%、至少60%、至少70%、至少80%、至少90%、至少95%或基本上100%。然后比较在相应的核苷酸位置处的核苷酸。当第一序列中的位置由与第二序列中的相应位置相同的核苷酸占据时,则分子在该位置处是相同的。两个序列之间的同一性百分比是由序列共享的相同位置的数目的函数,考虑到缺口的数目以及每个缺口的长度,所述缺口为了两个序列的最佳比对而需要引入。两个序列之间的序列比较和同一性百分比的确定可使用数学算法来完成。例如,可使用Meyers和Miller(CABIOS,1989,4:11-17)的算法确定两个核苷酸序列之间的同一性百分比,所述算法已掺入ALIGN程序(版本2.0)内,使用PAM120权重残基表、缺口长度罚分12和缺口罚分4。可替代地,可使用GCG软件包中的GAP程序使用NWSgapdna.CMP矩阵来确定两个核苷酸序列之间的同一性百分比。各种其他序列比对程序是可用的并且可用于确定序列同一性,例如Clustal。
集成的活细胞密度:如本文使用的,术语“集成的活细胞密度”指经过培养过程活细胞的平均密度乘以培养已运行的时间量。假设所产生的多肽和/或蛋白质的量与经过培养过程存在的活细胞数目成比例,集成的活细胞密度是用于估计经过培养过程产生的多肽和/或蛋白质的量的有用工具。
经分离的:如本文使用的,术语“经分离的”指这样的物质和/或实体,其已(1)与最初产生时(无论是在自然界中和/或在实验环境中)它与之结合的至少一些组分分开,和/或(2)由人工产生、制备和/或制造。经分离的物质和/或实体可与约10%、约20%、约30%、约40%、约50%、约60%、约70%、约80%、约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%、约99%或多于约99%的它们最初与之结合的其他组分分开。在一些实施例中,经分离的试剂是约80%、约85%、约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%、约99%或多于约99%纯的。如本文使用的,如果物质基本上不含其他组分,则它是“纯的”。如本文使用的,经分离的物质和/或实体的纯度百分比的计算不应包括赋形剂(例如缓冲液、溶剂、水等)。
培养基:如本文使用的,术语“培养基”指含有滋养生长细胞的营养素的溶液。通常,这些溶液提供细胞的最小生长和/或存活所需的必需和非必需氨基酸、维生素、能源、脂质和微量元素。该溶液还可含有使生长和/或存活增强到最低速率以上的组分,包括激素和生长因子。在一些实施例中,将培养基配制为对于细胞存活和增殖最佳的pH和盐浓度。在一些实施例中,培养基可为“化学成分确定的培养基”-不含蛋白质、水解产物或未知组成的组分的无血清培养基。在一些实施例中,化学成分确定的培养基不含动物来源的组分,并且培养基内的所有组分都具有已知的化学结构。在一些实施例中,培养基可为“基于血清的培养基”-已补充有动物来源的组分例如但不限于胎牛血清、马血清、山羊血清、驴血清和/或其组合的培养基。
核酸:如本文使用的,术语“核酸”在其最广泛的意义上指掺入或可掺入寡核苷酸链内的化合物和/或物质。在一些实施例中,核酸是经由磷酸二酯键掺入或可掺入寡核苷酸链内的化合物和/或物质。在一些实施例中,“核酸”指个别核酸残基(例如核苷酸和/或核苷)。在一些实施例中,“核酸”指包含个别核酸残基的寡核苷酸链。如本文使用的,术语“寡核苷酸”和“多核苷酸”可互换使用。在一些实施例中,“核酸”涵盖RNA以及单链和/或双链DNA和/或cDNA。此外,术语“核酸”、“DNA”、“RNA”和/或类似术语包括核酸类似物,即具有除磷酸二酯主链外的类似物。例如,本领域已知且在主链中具有肽键代替磷酸二酯键的所谓“肽核酸”被视为在本发明的范围内。术语“编码氨基酸序列的核苷酸序列”包括其为彼此的简并形式和/或编码相同氨基酸序列的所有核苷酸序列。编码蛋白质和/或RNA的核苷酸序列可包括内含子。核酸可从天然源纯化,使用重组表达系统产生并且任选纯化,化学合成等。适当时,例如在化学合成分子的情况下,核酸可包含核苷类似物,例如具有化学修饰的碱基或糖、主链修饰等的类似物。除非另有说明,否则核酸序列以5'至3'方向呈现。术语“核酸片段”在本文中用于指其为更长核酸序列的一部分的核酸序列。在许多实施例中,核酸片段包含至少3、4、5、6、7、8、9、10个或更多个残基。在一些实施例中,核酸是或包含天然核苷(例如腺苷、胸苷、鸟苷、胞苷、尿苷、脱氧腺苷、脱氧胸苷、脱氧鸟苷和脱氧胞苷);核苷类似物(例如2-氨基腺苷、2-硫代胸苷、肌苷、吡咯并嘧啶、3-甲基腺苷、5-甲基胞苷、C-5丙炔基-胞苷、C-5丙炔基-尿苷、2-氨基腺苷、C5-溴尿苷、C5-氟尿苷、C5-碘尿苷、C5-丙炔基-尿苷、C5-丙炔基-胞苷、C5-甲基胞苷、2-氨基腺苷、7-脱氮腺苷、7-脱氮鸟苷、8-氧代腺苷、8-氧代鸟苷、O(6)-甲基鸟嘌呤、和2-硫代胞苷);化学修饰的碱基;生物学修饰的碱基(例如甲基化碱基);嵌入碱基;经修饰的糖(例如2'-氟核糖、核糖、2'-脱氧核糖、阿拉伯糖和己糖);和/或经修饰的磷酸酯基团(例如硫代磷酸酯和5'-N-亚磷酰胺键)。在一些实施例中,本发明具体涉及“未修饰的核酸”,意指未被化学修饰以便促进或实现递送的核酸(例如多核苷酸和残基,包括核苷酸和/或核苷)。
灌注过程:如本文使用的,术语“灌注过程”指培养细胞的方法,其中在培养过程开始之后连续或半连续地向培养物提供另外的组分。所提供的组分通常包含在培养过程期间已耗尽的用于细胞的营养补充剂。通常在连续或半连续的基础上收获培养基中的细胞和/或组分的一部分,并且任选进行纯化。通常,涉及灌注过程的细胞培养过程被称为“灌注培养”。通常,在灌注过程期间在新鲜培养基中提供营养补充剂。在一些实施例中,新鲜培养基可与细胞培养过程中使用的基础培养基相同或相似。在一些实施例中,新鲜培养基可不同于基础培养基,但含有所需营养补充剂。在一些实施例中,新鲜培养基是化学成分确定的培养基。
接种:如本文使用的,术语“接种”指将细胞培养物提供给用于大规模细胞培养生产的生物反应器或另一容器的过程。在一些实施例中,使用“种子培养物,”其中在接种之前,细胞已在更小的细胞培养容器即组织培养瓶、组织培养板、组织培养滚瓶等中繁殖。可替代地,在一些实施例中,细胞可能已被冷冻且在将它们提供给生物反应器或容器之前立即解冻。该术语指任何数目的细胞,包括单个细胞。
受试者:如本文使用的,术语“受试者”意指任何哺乳动物,包括人。在本发明的某些实施例中,受试者是成人、青少年或婴儿。本发明还考虑了药物组合物的施用和/或宫内治疗方法的执行。
载体:如本文使用的,“载体”指能够转运它与之结合的另一种核酸的核酸分子。在一些实施例中,载体能够在宿主细胞例如真核和/或原核细胞中染色体外复制和/或表达它们与之连接的核酸。能够指导可操作地连接的基因的表达的载体在本文中称为“表达载体”。
活细胞密度:如本文使用的,术语“活细胞密度”指活细胞数目/单位体积。
具体实施方式
本发明尤其提供了使用SUMO-鸟苷酰转移酶融合蛋白用于大规模生产加帽的mRNA的方法和组合物。
在下述小节中进一步详细描述本发明的各个方面。小节的使用并不意欲限制本发明。每个小节可适用于本发明的任何方面。在本专利申请中,除非另有说明,否则“或”的使用意指“和/或”。
SUMO-鸟苷酰转移酶融合蛋白
小泛素样改性剂(SUMO)
如本文使用的,SUMO标签是可取代SUMO蛋白的至少部分活性的任何蛋白质或蛋白质的一部分。
SUMO蛋白是与其他蛋白质共价附着和脱离以便修饰这些蛋白质的功能的小蛋白质。用SUMO蛋白修饰蛋白质是涉及各种细胞过程的翻译后修饰,所述细胞过程例如核-胞质转运、转录调节、细胞凋亡、蛋白质稳定性、对应激的应答和通过细胞周期的进展。脊椎动物中存在至少4种SUMO旁系同源物,指定为SUMO-1、SUMO-2、SUMO-3和SUMO-4。SUMO-2和SUMO-3在结构和功能上非常相似,并且与SUMO-1不同。表1中显示了跨越通常的野生型或天然存在的SUMO-3蛋白的氨基酸3-92的氨基酸序列(SEQ ID NO:1)。另外,编码SUMO-3蛋白的密码子优化的DNA序列也在表1中作为SEQ ID NO:5提供。
表1.小泛素样改性剂
因此,在一些实施例中,SUMO蛋白是人SUMO-3蛋白(SEQ ID NO:1)。在一些实施例中,SUMO蛋白可为另一种SUMO旁系同源物,例如SUMO-1、SUMO-2或SUMO-4。在一些实施例中,合适的替代蛋白可为人SUMO-3蛋白的同源物或类似物。例如,SUMO-3蛋白的同源物或类似物可为与野生型或天然存在的SUMO-3蛋白(例如SEQ ID NO:1)相比,含有一个或多个氨基酸取代、缺失和/或插入,同时保留了基本SUMO-3蛋白活性的经修饰的SUMO-3蛋白。因此,在一些实施例中,适合于本发明的酶与野生型或天然存在的SUMO-3蛋白(SEQ ID NO:1)基本上同源。在一些实施例中,适合于本发明的酶具有与SEQ ID NO:1至少50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%相同或更相同的氨基酸序列。在一些实施例中,适合于本发明的酶与野生型或天然存在的SUMO-3蛋白(SEQ ID NO:1)基本上相同。在一些实施例中,适合于本发明的蛋白含有SUMO蛋白的片段或一部分。在一些实施例中,SUMO蛋白包含人SUMO-1、人SUMO-2、人SUMO-3、拟南芥(Arabidopsis Zhalania)SUMO-l至SUMO-8中的任何一种、番茄SUMO、非洲爪蟾(Xenopuslaevis)SUMO-l至SUMO-3中的任何一种、黑腹果蝇(Drosophila melanogasler)Smt3、秀丽隐杆线虫(Caenorhabdilis elegans)SMO-1、粟酒裂殖酵母(Schizosaccharomycespombe)Pmt3、疟原虫恶性疟原虫(Plasmodium falciparum)SUMO、霉菌构巢曲霉(Aspergillusnidulans)SUMO、其等价物、其同源物或其组合。
在一些实施例中,SUMO蛋白由源自生物的核酸编码,所述生物选自人、小鼠、昆虫、植物、酵母和其他真核生物。在一些实施例中,SUMO蛋白由源自生物的核酸编码,所述生物选自智人(Homo sapiens)、拟南芥、番茄、非洲爪蟾、黑腹果蝇、秀丽隐杆线虫、粟酒裂殖酵母、恶性疟原虫或构巢曲霉。在一些实施例中,适合于本发明的核酸具有与SEQ ID NO:
5至少50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%相同或更相同的序列。在一些实施例中,适合于本发明的核酸与编码野生型或天然存在的SUMO-3蛋白的核酸(SEQ ID NO:5)基本上相同。
鸟苷酰转移酶(GT)
如本文使用的,GT蛋白是可取代天然存在的鸟苷酰转移酶(GT)蛋白的至少部分活性的任何蛋白质或蛋白质的一部分。如本文使用的,术语“GT蛋白”和“GT酶”和语法等价物可互换使用。
GT是源自痘苗病毒系统的酶,其促进鸟苷酰分子转移至信使RNA分子的5'末端和鸟苷酰分子的甲基化。这个过程被称为mRNA加帽,受到高度调节,并且对于能够在蛋白质合成过程中经历翻译的稳定和成熟mRNA的产生很重要。GT酶包含包括“大亚基”(D1,约97kDa)和“小亚基”(D12,约33kDa)的异源二聚体。GT提供三种酶功能:磷酸酶活性(mRNA的新生5'三磷酸切割为二磷酸)、鸟苷酰转移酶活性(GTP分子掺入mRNA部分的5'末端)和甲基化活性(在鸟苷酰碱基的N7位置处掺入甲基)。表2显示了通常的野生型或天然存在的GT蛋白的大亚基(SEQ ID NO:6)和小亚基(SEQ ID NO:7)的氨基酸序列。另外,编码GT的大亚基和小亚基的密码子优化的DNA序列还在表2中分别作为SEQ ID NO:2和SEQ ID NO:3提供。
表2.鸟苷酰转移酶
因此,在一些实施例中,GT酶是包含大亚基和小亚基(分别为SEQ ID NO:6和SEQID NO:7)的异源二聚体。在一些实施例中,本发明的GT酶可为GT大亚基和小亚基中的一个或另一个的同源物或类似物。例如,GT蛋白的同源物或类似物可为与SEQ ID NO:6和/或SEQID NO:7相比,含有一个或多个氨基酸取代、缺失和/或插入,同时保留了基本GT蛋白活性的经修饰的GT蛋白。因此,在一些实施例中,适合于本发明的酶与GT蛋白大亚基和小亚基(SEQID NO:6和SEQ ID NO:7)基本上同源。在一些实施例中,适合于本发明的酶具有与SEQ IDNO:6至少50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%相同或更相同的氨基酸序列。在一些实施例中,适合于本发明的酶具有与SEQ ID NO:7至少50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%相同或更相同的氨基酸序列。在一些实施例中,适合于本发明的酶与GT的大亚基和小亚基(SEQ ID NO:6和SEQ ID NO:7)基本上相同。在一些实施例中,适合于本发明的酶含有GT蛋白的片段或一部分。
在一些实施例中,GT蛋白由源自病毒的核酸编码,所述病毒选自痘苗病毒、兔痘病毒、牛痘病毒、沙鼠痘病毒、猴痘病毒、重型天花病毒、骆驼痘病毒、鼠痘病毒、瓦瑞奥拉轻型天花病毒、正痘病毒、浣熊痘病毒、臭鼬痘病毒、田鼠痘病毒、约卡痘病毒(Yoka poxvirus)、猪痘病毒、雅巴猴瘤病毒、鹿痘病毒、粘液瘤病毒、塔纳痘病毒、山羊痘病毒、兔纤维瘤病毒、结节性皮肤病病毒、绵羊痘病毒、Eptesipox病毒、松鼠痘病毒、传染性软疣病毒、科蒂亚病毒、口疮病毒、牛流行性口炎病毒、假牛痘病毒、金丝雀痘病毒、鸽痘病毒(Pidgeonpox virus)、企鹅痘病毒和禽痘病毒。在一些实施例中,适合于本发明的核酸具有与SEQ ID NO:2至少50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%相同或更相同的序列。在一些实施例中,适合于本发明的核酸具有与SEQ ID NO:3至少50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%相同或更相同的序列。在一些实施例中,适合于本发明的核酸与编码GT蛋白的核酸(SEQ ID NO:2和SEQ ID NO:3)基本上相同。
SUMO-GT融合物
如本文使用的,SUMO-GT融合蛋白是包含与鸟苷酰转移酶(GT)蛋白共价连接的SUMO蛋白的任何蛋白质或蛋白质的一部分,其中所述融合蛋白可取代天然存在的鸟苷酰转移酶(GT)蛋白的至少部分活性。如本文使用的,术语“SUMO-GT融合蛋白”和“SUMO-GT融合酶”和语法等价物可互换使用。SUMO和GT大亚基的融合物的示例性氨基酸序列(SEQ ID NO:8)显示于表3中。另外,编码SUMO和GT大亚基的融合物的示例性DNA序列也作为SEQ ID NO:4在表3中提供。
表3.SUMO-GT融合物
在一些实施例中,SUMO-GT融合蛋白包含SEQ ID NO:8。在一些实施例中,SUMO-GT融合蛋白是包含SEQ ID NO:8和SEQ ID NO:7的异源二聚体。在一些实施例中,本发明的GT酶可为GT大亚基和小亚基中的一个或另一个的同源物或类似物。例如,SUMO-GT融合蛋白的同源物或类似物可为与SEQ ID NO:8和/或SEQ ID NO:7相比,含有一个或多个氨基酸取代、缺失和/或插入,同时保留了基本GT蛋白活性的经修饰的SUMO-GT融合蛋白。因此,在一些实施例中,适合于本发明的SUMO-GT融合蛋白与包含GT小亚基(SEQ ID NO:7)和SUMO与GT大亚基的融合物(SEQ ID NO:8)的异源二聚体基本上同源。在一些实施例中,适合于本发明的酶具有与SEQ ID NO:8和SEQ ID NO:7至少50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%相同或更相同的氨基酸序列。在一些实施例中,适合于本发明的酶与包含GT小亚基(SEQ ID NO:7)和SUMO与GT大亚基的融合物(SEQ ID NO:8)的异源二聚体基本上相同。在一些实施例中,适合于本发明的酶含有与SUMO蛋白共价结合的GT蛋白的片段或一部分。
SUMO-GT融合蛋白的产生
宿主细胞
如本文使用的,术语“宿主细胞”指可用于产生SUMO-GT融合蛋白的细胞。特别地,宿主细胞适合于大规模生产SUMO-GT融合蛋白。在一些实施例中,宿主细胞能够以为或大于约5皮克/细胞/天(例如大于约10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95或100皮克/细胞/天)的量生产SUMO-GT融合蛋白。在一些实施例中,宿主细胞能够以范围为约5-100皮克/细胞/天(例如约5-90皮克/细胞/天、约5-80皮克/细胞/天、约5-70皮克/细胞/天、约5-60皮克/细胞/天、约5-50皮克/细胞/天、约5-40皮克/细胞/天、约5-30皮克/细胞/天、约10-90皮克/细胞/天、约10-80皮克/细胞/天、约10-70皮克/细胞/天、约10-60皮克/细胞/天、约10-50皮克/细胞/天、约10-40皮克/细胞/天、约10-30皮克/细胞/天、约20-90皮克/细胞/天、约20-80皮克/细胞/天、约20-70皮克/细胞/天、约20-60皮克/细胞/天、约20-50皮克/细胞/天、约20-40皮克/细胞/天、约20-30皮克/细胞/天)的量生产SUMO-GT融合蛋白。
合适的宿主细胞可来自各种生物,其包括但不限于细菌、酵母、昆虫、植物、鸟类(例如禽类系统)、两栖类动物和哺乳动物。在一些实施例中,宿主细胞是非哺乳动物细胞。适合于本发明的非哺乳动物宿主细胞的非限制性例子包括源自下述的细胞和细胞系:关于细菌的大肠杆菌(Escherichia coli)、鼠伤寒沙门氏菌(Salmonella typhimurium)、枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus lichenifonnis)、脆弱拟杆菌(Bacteroidesfragilis)、产气荚膜梭菌(Clostridia perfringens)、艰难梭菌(Clostridia difficile);关于酵母的巴斯德毕赤酵母(Pichia pastoris)、甲醇毕赤酵母(Pichia methanolica)、安格斯毕赤酵母(Pichia angusta)、粟酒裂殖酵母、酿酒酵母(Saccharomyces cerevisiae)和解脂耶氏酵母(Yarrowia lipolytica);关于昆虫的草地贪夜蛾(Sodoptera frugiperda)、粉纹夜蛾(Trichoplusis ni)、黑腹果蝇和烟草天蛾(Manduca sexta);和来自两栖类动物的非洲爪蟾。
在一些实施例中,宿主细胞是哺乳动物细胞。对细胞培养和对多肽表达敏感的任何哺乳动物细胞均可根据本发明用作宿主细胞。可根据本发明使用的哺乳动物细胞的非限制性例子包括人胚肾293细胞(HEK293)、HeLa细胞;BALB/c小鼠骨髓瘤细胞系(NSO/l,ECACC编号:85110503);人成视网膜细胞(PER.C6(CruCell,Leiden,荷兰));通过SV40转化的猴肾CV1系(COS-7,ATCC CRL 1651);人纤维肉瘤细胞系(例如HT-1080);人胚胎肾系(293细胞或亚克隆用于在悬浮培养中生长的293细胞,Graham等人,J.Gen Virol.,36:59(1977));幼仓鼠肾细胞(BHK,ATCC CCL 10);中国仓鼠卵巢细胞+/-DHFR(CHO,Urlaub和Chasin,Proc.Natl.Acad.Sci.USA,77:4216(1980));小鼠滋养细胞(TM4,Mather,Biol.Reprod.,23:243-251(1980));猴肾细胞(CV1 ATCC CCL 70);非洲绿猴肾细胞(VERO-76,ATCC CRL-1587);人宫颈癌细胞(HeLa,ATCC CCL 2);犬肾细胞(MDCK,ATCC CCL 34);水牛鼠肝细胞(BRL3A,ATCC CRL 1442);人肺细胞(W138,ATCC CCL 75);人肝细胞(Hep G2,HB 8065);小鼠乳房肿瘤(MMT 060562,ATCC CCL51);TRI细胞(Mather等人,Annals N.Y.Acad.Sci.,383:44-68(1982));MRC 5细胞;FS4细胞;人肝癌系(Hep G2),人细胞系CAP和AGE1.HN,以及Glycotope的实验对象组。
另外,任何数目的可用杂交瘤细胞系可根据本发明利用。本领域技术人员应了解,杂交瘤细胞系可能具有不同的营养需求和/或可能需要不同的培养条件,用于最佳生长和多肽或蛋白质表达,并且将能够根据需要修改条件。
表达载体
各种核酸构建体可用于在宿主细胞中表达本文所述的SUMO-GT融合蛋白。除SUMO-GT融合蛋白编码序列(也称为SUMO-GT融合转基因)之外,合适的载体构建体通常包括调节序列、基因控制序列、启动子、非编码序列和/或用于蛋白质表达以及任选的用于构建体复制的其他适当序列。通常,编码区与这些核酸组分中的一种或多种可操作地连接。
“调节序列”通常指位于编码序列的上游(5'非编码序列)、编码序列的内部或编码序列的下游(3'非编码序列),并且影响相关编码序列的转录、RNA加工或稳定性或翻译的核苷酸序列。调节序列可包括启动子、增强子、5'非翻译序列、翻译前导序列、内含子和3'非翻译序列例如聚腺苷酸化识别序列。有时,“调节序列”也称为“基因控制序列”。
“启动子”通常指能够控制编码序列或功能性RNA表达的核苷酸序列。一般而言,编码序列位于启动子序列的3'。启动子序列由近端和更远端的上游元件组成,后面一种元件通常称为增强子。相应地,“增强子”是可刺激启动子活性的核苷酸序列,并且可为启动子的固有元件或被插入以增强启动子的水平或组织特异性的异源元件。启动子可整体源自天然基因,或者由源自自然界中发现的不同启动子的不同元件组成,或者甚至包含合成的核苷酸区段。本领域技术人员应理解,不同的启动子可指导基因在不同组织或细胞类型中、或在不同发育阶段、或响应不同环境条件的表达。
“3'非编码序列”通常指位于编码序列的下游的核苷酸序列,并且包括聚腺苷酸化识别序列和编码能够影响mRNA加工或基因表达的调节信号的其他序列。聚腺苷酸化信号的特征通常在于影响聚腺苷酸束对mRNA前体的3'末端的添加。
“翻译前导序列”或“5'非编码序列”通常指位于基因的启动子序列和编码序列之间的核苷酸序列。翻译前导序列存在于翻译起始序列上游的完全加工的mRNA中。翻译前导序列可影响初级转录物至mRNA的加工、mRNA稳定性或翻译效率。
通常,术语“可操作地连接”指在单个核酸片段上两个或更多个核酸片段的结合,使得一个的功能受另一个的影响。例如,当启动子能够影响编码序列的表达(即编码序列处于启动子的转录控制之下)时,启动子与该编码序列可操作地连接。编码序列可以在有义或反义方向上与调节序列可操作地连接。
转基因的编码区可包括一个或多个沉默突变,以优化对于特定细胞类型的密码子使用。例如,SUMO-GT融合转基因的密码子可对于在细菌细胞中表达进行优化。在一些实施例中,SUMO-GT融合转基因的密码子可对于在大肠杆菌细胞中表达进行优化。在一些实施例中,SUMO-GT融合转基因的密码子可对于在哺乳动物细胞中表达进行优化。在一些实施例中,SUMO-GT融合转基因的密码子可对于在人细胞中表达进行优化。
任选地,构建体可含有另外的组分,例如下述中的一种或多种:剪接位点、增强子序列、在适当启动子的控制下的可选择标志物基因、在适当启动子的控制下的可扩增标志物基因和基质附着区(MAR)或本领域已知的增强它插入其中的区域的表达的其他元件。
一旦转染或转导到宿主细胞中,合适的载体就可染色体外(游离体)表达或整合到宿主细胞的基因组内。
在一些实施例中,整合到细胞的基因组内的DNA构建体仅需要包括转基因核酸序列。在所述情况下,转基因的表达通常由在整合位点处的调节序列控制。任选地,它可包括本文所述的另外的各种调节序列。
培养基和条件
如本文使用的,术语“培养基(medium)”和“培养基(culture medium)”指含有适合于在体外维持和/或生长细胞的营养素的一般类别的溶液。通常,培养基溶液提供(但不限于)细胞对于至少最小生长和/或存活所需的必需和非必需氨基酸、维生素、能源、脂质和微量元素。在其他实施例中,培养基可含有源自本领域已知的任何来源或方法的氨基酸,其包括但不限于源自单一氨基酸添加或蛋白胨或蛋白质水解产物添加(包括动物或植物来源)的氨基酸。维生素例如但不限于生物素、泛酸盐、氯化胆碱、叶酸、肌醇、烟酰胺、吡哆醇、核黄素、维生素B12、硫胺素、腐胺和/或其组合。盐例如但不限于CaCl2、KCl、MgCl2、NaCl、磷酸二氢钠、磷酸氢二钠、亚硒酸钠、CuSO4、ZnCl2和/或其组合。脂肪酸例如但不限于花生四烯酸、亚油酸、油酸、月桂酸、肉豆蔻酸、以及甲基-β-环糊精和/或其组合。在一些实施例中,培养基包含另外的组分如葡萄糖、谷氨酰胺、丙酮酸钠、胰岛素或乙醇胺、保护剂如PluronicF68。在一些实施例中,培养基还可含有使生长和/或存活增强到最小速率以上的组分,包括激素和生长因子。培养基还可包含一种或多种缓冲剂。缓冲剂可设计和/或选择为将培养物维持在特定的pH(例如生理pH(例如pH 6.8至pH 7.4))下。适合于培养细胞的各种缓冲液是本领域已知的,并且可用于这些方法中。合适的缓冲液(例如碳酸氢盐缓冲液、HEPES缓冲液、Good's缓冲液等)是不管与细胞呼吸相关的二氧化碳浓度的变化而具有维持生理pH的能力和效率的那些缓冲液。该溶液优选配制为对于细胞存活和增殖最佳的pH和盐浓度。
在一些实施例中,培养基可为化学成分确定的培养基。如本文使用的,术语“化学成分确定的营养培养基”指基本上所有化学组分都是已知的培养基。在一些实施例中,化学成分确定的营养培养基不含动物来源的组分。在一些情况下,化学成分确定的培养基包含一种或多种蛋白质(例如蛋白质生长因子或细胞因子)。在一些情况下,化学成分确定的营养培养基包含一种或多种蛋白质水解产物。在其他情况下,化学成分确定的营养培养基是不含蛋白质的培养基,即不含蛋白质、水解产物或未知组成的组分的无血清培养基。
通常,化学成分确定的培养基可通过将以预定重量或摩尔百分比或比率的各种个别组分(例如必需和非必需氨基酸、维生素、能源、脂质、盐、缓冲剂和微量元素)组合来制备。示例性的无血清培养基,特别是化学成分确定的培养基在美国公开号2006/0148074中描述,所述美国公开的公开内容在此以引用的方式并入。
在一些实施例中,适合于本发明的化学成分确定的培养基是商购可得的培养基,例如但不限于Terrific Broth、Cinnabar、2xYT或LB。在一些实施例中,适合于本发明的化学成分确定的培养基是一种或多种商购可得的化学成分确定的培养基的混合物。在各种实施例中,合适的培养基是二、三、四、五、六、七、八、九、十种或更多种商业上可获得的化学成分确定的培养基的混合物。在一些实施例中,每种个别商购可得的化学成分确定的培养基(例如如本文所述的那些)构成混合物的按重量计的1%、2.5%、5%、7.5%、10%、12.5%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或更多。每种个别组分培养基之间的比率可通过混合物中存在的相对重量百分比来确定。在一些实施例中,通过添加IPTG阻遏启动子来增加蛋白质表达。
在一些实施例中,化学成分确定的培养基可通过一种或多种动物来源的组分补充。这样的动物来源的组分包括但不限于胎牛血清、马血清、山羊血清、驴血清、人血清和血清来源的蛋白质例如白蛋白(例如牛血清白蛋白或人血清白蛋白)。
本发明提供了以大规模生产SUMO-GT融合蛋白的方法。用于生产目的融合多肽的通常大规模程序包括分批培养和补料分批培养。分批培养方法通常包括用特定细胞密度的种子培养物接种大规模生产培养物,在有助于细胞生长、活力和/或生产力的条件下(例如合适的培养基、pH和温度)使细胞生长,当细胞达到特定细胞密度时收获培养物,并且纯化所表达的多肽。补料分批培养程序包括用在细胞生长期间消耗的营养素和其他组分补充分批培养物的另外的一个或多个步骤。在一些实施例中,根据本发明的大规模生产方法使用补料分批培养系统。
所表达的SUMO-GT融合蛋白的纯化
各种方法可用于纯化或分离根据本文所述的各种方法产生的SUMO-GT融合蛋白。在一些实施例中,所表达的SUMO-GT融合蛋白分泌到培养基内,并且因此可通过例如离心或过滤去除细胞和其他固体,作为纯化过程中的第一步。可替代地或另外地,所表达的SUMO-GT融合蛋白结合到宿主细胞的表面。在该实施例中,表达多肽或蛋白质的宿主细胞(例如细菌细胞)被裂解用于纯化。宿主细胞(例如细菌细胞)的裂解可通过本领域普通技术人员众所周知的任何数目的手段来实现,包括通过玻璃珠的物理破碎和暴露于高pH条件。
可通过标准方法分离且纯化SUMO-GT融合蛋白,所述标准方法包括但不限于层析(例如离子交换、亲和力、尺寸排阻和羟磷灰石层析)、凝胶过滤、离心或差异溶解度、乙醇沉淀或通过用于纯化蛋白质的任何其他可用技术(参见例如Scopes,Protein PurificationPrinciples and Practice第2版,Springer-Verlag,New York,1987;Higgins,S.J.和Hames,B.D(编辑),Protein Expression:A Practical Approach,Oxford Univ Press,1999;和Deutscher,M.P.,Simon,M.I.,Abelson,J.N.(编辑),Guide to ProteinPurification:Methods in Enzymology(Methods in Enzymology Series,第182卷),Academic Press,1997,所有所述参考文献都以引用的方式并入本文)。特别地对于免疫亲和层析,蛋白质可通过将其与包含抗体的亲和柱结合来分离,该抗体针对该蛋白质产生并且附着到固定支持物。蛋白酶抑制剂例如苯基甲基磺酰氟(PMSF)、亮肽素、胃酶抑素或抑肽酶可在任何阶段或所有阶段时添加,以便减少或消除在纯化过程期间多肽或蛋白质的降解。当细胞必须裂解以便分离和纯化所表达的多肽或蛋白质时,蛋白酶抑制剂是特别需要的。
溶解度
各种方法可用于确定蛋白质在表达系统中的溶解度。在示例性方法中,将细菌向下旋转并且重悬浮于含有1%IGEPAL和蛋白酶抑制剂的温和裂解缓冲液中。通过反复冻融细菌来支持裂解。通过离心分离可溶性和不溶性级分。为了确定重组蛋白质的总量,将相同体积的细菌培养物向下旋转,并且在相同量的含有1%IGEPAL和0.1%SDS的裂解缓冲液中裂解。通过SDS-PAGE,必要时伴随蛋白质印迹,来分析可溶性蛋白质和总蛋白质。在一些实施例中,表达系统是大肠杆菌。在一些实施例中,当GT已作为融合蛋白产生时,GT的溶解度得到改善。在一些实施例中,融合蛋白是SUMO-GT融合蛋白。在一些实施例中,与非融合GT蛋白相比,SUMO-GT融合蛋白具有增加的溶解度。在一些实施例中,在SUMO-GT融合蛋白的摇瓶生产期间,观察到与非融合GT蛋白相比SUMO-GT融合蛋白增加的溶解度。在一些实施例中,在SUMO-GT融合蛋白的发酵生产期间,观察到与非融合GT蛋白相比SUMO-GT融合蛋白增加的溶解度。
SUMO-GT融合物在mRNA加帽中的用途
加帽的mRNA的产生
根据本发明,本文所述的SUMO-GT融合蛋白可用于通过体外转录来产生加帽的mRNA。本领域可获得各种体外转录测定并且可用于实践本发明。例如,体外转录最初由Krieg和Melton(Methods Enzymol.,1987,155:397-415)开发,用于使用RNA噬菌体聚合酶合成RNA。通常,这些反应至少包括噬菌体RNA聚合酶(T7、T3或SP6)、含有噬菌体聚合酶启动子的DNA模板、核苷酸(ATP、CTP、GTP和UTP)和含有镁盐的缓冲液。可通过增加核苷酸浓度、调节镁浓度和通过包括无机焦磷酸酶来最佳化RNA合成产率(美国专利号5,256,555;Gurevich等人,Anal.Biochem.195:207-213(1991);Sampson,J.R.和Uhlenbeck,O.C.,Proc.Natl.Acad.Sci.USA.85,1033-1037(1988);Wyatt,J.R.等人,Biotechniques,11:764-769(1991))。在这些反应中合成的RNA的特征通常在于在核糖的5'位置处具有三磷酸的5'末端核苷酸。通常,取决于所使用的RNA聚合酶和启动子组合,该核苷酸是鸟苷,尽管它可为腺苷(参见例如Coleman,T.M.等人,Nucleic Acids Res.,32:e14(2004))。在这些反应中,所有四种核苷酸通常以等摩尔浓度包含,并且它们中无一是限制性的。
本发明的一些实施例是分批反应—即将所有组分组合,然后在约37℃下温育以促进RNA的聚合,直至反应终止。通常,为了方便起见使用分批反应,并且从这些反应中获得尽可能多的需要的RNA用于其实验。在一些实施例中,“补料分批”系统(参见例如JeffreyA.Kern,Batch and Fed-batch Strategies for Large-scale Production of RNA by inVitro Transaction(University of Colorado)(1997))用于增加体外转录反应的效率。将所有组分组合,然后随着时间过去添加另外量的一些试剂,例如核苷酸和镁,以尝试维持恒定的反应条件。另外,在一些实施例中,通过随着时间过去监测pH并且根据需要添加KOH,可将反应的pH保持在7.4下。
为了通过体外转录合成加帽的RNA,将帽类似物(例如N-7甲基GpppG;即,m7GpppG)包括在转录反应中。在一些实施例中,帽类似物将在5'末端处通过鸟苷酰转移酶掺入。在一些实施例中,鸟苷酰转移酶是融合蛋白。在一些实施例中,当鸟苷酰转移酶与SUMO蛋白共价连接时,形成鸟苷酰转移酶融合蛋白。在一些实施例中,帽类似物将仅在5'末端处掺入,因为它不具有5'三磷酸。在使用T7、T3和SP6 RNA聚合酶的一些实施例中,它们各自启动子的+1核苷酸通常是G残基,并且如果GTP和m7GpppG两者在转录反应中均以相等浓度存在,则它们各自具有在+1位置处掺入的相等机会。在一些实施例中,m7GpppG以是GTP几倍高的浓度存在于这些反应中,以增加转录物具有5'帽的机会。在一些实施例中,根据制造商的说明书使用mMESSAGE试剂盒(目录#1344,Ambion,Inc.),其中推荐帽与GTP的比率为4:1(6mM:1.5mM)。在一些实施例中,随着反应中帽类似物与GTP的比率增加,加帽的RNA与未加帽的RNA的比率按比例增加。加帽效率的考虑必须与产率的考虑相平衡。在转录反应中增加帽类似物与GTP的比率产生较低的总RNA产率,因为当帽和GTP的总浓度保持恒定时,GTP的浓度变得是限制性的。因此,最终的RNA产率取决于GTP浓度,这对于转录物的延伸是必需的。其他核苷酸(ATP、CTP、UTP)以过量存在。
在特定实施例中,mRNA通过由编码选择基因的质粒DNA模板体外转录合成。在一些实施例中,体外转录包括经由鸟苷酰转移酶通过GTP的酶促缀合添加5'帽结构Cap1(图1B),所述Cap1在碱基1的核糖环的2'OH基处具有2'-O-甲基残基。在一些实施例中,体外转录包括经由鸟苷酰转移酶通过GTP的酶促缀合添加5'帽结构Cap0(图1A),所述Cap0缺少2'-O-甲基残基。在一些实施例中,体外转录包括经由鸟苷酰转移酶通过GTP的酶促缀合添加本文公开的任何帽结构的5'帽。
加帽效率
本发明显著增加了加帽效率。在一些实施例中,在体外加帽测定中使用SUMO-GT融合蛋白导致至少约70%、75%、80%、85%、90%、95%、96%、97%、98%或99%的加帽的mRNA。在一些实施例中,在体外加帽测定中使用SUMO-GT融合蛋白导致基本上100%的加帽的mRNA。在一些实施例中,与使用非融合GT蛋白但在其他方面相同的条件下的对照测定相比,在体外加帽测定中使用SUMO-GT融合蛋白导致mRNA加帽效率增加至少约20%、30%、40%、50%、60%、70%、80%、90%、95%、1倍、1.5倍、2倍、2.5倍、3倍、3.5倍、4倍、4.5倍或5倍。
另外,本发明允许以高效率大规模生产加帽的mRNA。在一些实施例中,以为或大于1克、5克、10克、15克、20克、25克、30克、35克、40克、45克、50克、75克、100克、150克、200克、250克、300克、350克、400克、450克、500克、550克、600克、650克、700克、750克、800克、850克、900克、950克、1kg、2.5kg、5kg、7.5kg、10kg、25kg、50kg、75kg或100kg/分批的规模生产加帽的mRNA。
估计加帽效率的方法是本领域已知的。例如,T7 RNA聚合酶可与帽二核苷酸、所有四种核糖核苷酸三磷酸、[α-32P]GTP和短DNA模板一起温育,其中G是启动子后指定的第一个核糖核苷酸(参见Grudzien,E.等人“Novel cap analogs for in vitro synthesis ofmRNA with high transla tion efficiency”,RNA,10:1479-1487(2004))。在G残基的5'侧上的任何核苷酸在RNase T2消化后通过最近邻转移获得32P标记的3'-磷酸酯基团。阴离子交换层析然后用于从5'-末端产物分辨来源于RNA中的内部位置的标记的核苷3'-单磷酸酯。5'-末端产物具有两种类型。未加帽的RNA产生标记的鸟苷5'-三磷酸3'-单磷酸酯(p3Gp*;其中*指示标记的磷酸酯基团)。根据所使用的帽类似物的性质,加帽的RNA产生各种5'-末端结构(当帽类似物是m7Gp3G时,为m7Gp3Gp*和Gp3m7Gp*)。
在WO 2014/152673中提供了直接定量样品(例如来自体外合成反应的代表性等分样品)中的mRNA加帽效率的改进方法,所述公开以引用的方式并入本文。一些实施例包括在允许帽特异性结合物质和加帽的mRNA之间形成复合物的条件下,使用帽特异性结合物质。在帽特异性结合物质和加帽的mRNA之间形成复合物允许复合物(即,加帽的mRNA)的量相对于加帽产物的阳性对照或未加帽产物的阴性对照的定量测定。换言之,结合指示样品中加帽的mRNA靶的量和样品由其衍生的反应中的加帽效率。因此,在一些实施例中,定量确定复合物的量的步骤包括执行ELISA型测定,其中帽特异性结合物质是特异性结合mRNA帽的抗体或其他蛋白质。复合物形成可通过下述进行定量:添加对于帽特异性结合物质特异性的检测试剂(例如结合小鼠抗m7G抗体的山羊抗小鼠抗体),所述检测试剂产生与加帽的mRNA的量成正比的信号。本发明的实施例可用于定量广泛多样的RNA种类的加帽效率,所述RNA种类包括体外转录的mRNA、经分离的真核mRNA和病毒RNA。
在WO 2014/152659中提供了直接定量样品(例如来自体外合成反应的代表性等分样品)中的mRNA加帽效率的另外改进方法,所述公开以引用的方式并入本文。本发明的一些实施例包括定量mRNA加帽效率的层析方法。这些方法部分基于以下见解:酶促操作的多功能性可用于增加多核苷酸的层析分离的分辨率。因此,通过经由酶促操作放大层析分离的能力,本发明的实施例增加定量的效率、质量和流通量。例如,本文描述的层析方法不仅可定量加帽效率,它们还可提供关于帽的修饰(例如在特定帽位置处的甲基化状态)的信息。因此,本发明的实施例可同时定量加帽效率和帽修饰的效率(例如甲基化效率)。该定量提供了对蛋白质生产具有显著影响的mRNA药物产品的重要特征。
本发明通过参考下述实例将得到更全面地理解。然而,它们不应被理解为限制本发明的范围。所有文献引用均以引用的方式并入。
实例
实例1:SUMO-GT构建体设计
合成掺入小泛素样改性剂(SUMO)标签的新构建体,所述SUMO标签与鸟苷酰转移酶(GT)异源二聚体的大亚基部分共价连接且共表达。
小泛素样改性剂(SUMO)DNA:
GAAGAGAAACCGAAAGAGGGCGTTAAGACCGAGAATGACCACATTAACCTGAAGGTCGCTGGTCAAGATGGCAGCGTGGTGCAGTTTAAGATCAAGCGTCACACGCCGTTGAGCAAGCTGATGAAGGCTTACTGCGAGCGTCAGGGTCTGAGCATGCGTCAGATCCGCTTTCGTTTCGATGGCCAGCCGATCAATGAGACTGACACCCCAGCGCAACTGG(SEQ ID NO:5)
鸟苷酰转移酶(GT)大亚基DNA:
AGATGGAAGATGAAGATACCATCGACGTCTTTCAGCAACAGACCGGTGGTATGGATGCTAACGTCGTTAGCAGCAGCACCATTGCGACTTACATTGATGCACTGGCCAAAAACGCATCTGAGCTTGAGCAGCGCAGCACCGCCTACGAGATCAATAACGAATTGGAGCTGGTTTTCATTAAACCGCCGCTGATCACGCTGACGAACGTCGTGAACATTAGCACGATTCAAGAGAGCTTTATTCGTTTCACCGTTACCAATAAAGAAGGCGTGAAGATCCGTACCAAGATTCCGCTGAGCAAAGTGCATGGTCTGGACGTGAAAAATGTGCAGCTGGTTGATGCGATCGATAACATCGTGTGGGAGAAGAAATCTTTGGTCACGGAAAATCGTCTGCACAAGGAATGTCTGCTGCGTCTGTCAACCGAAGAACGCCACATCTTCCTGGACTACAAGAAGTATGGTTCCAGCATCCGTCTGGAACTGGTGAACCTGATTCAGGCAAAGACCAAGAACTTCACCATTGACTTCAAACTGAAGTATTTCCTGGGCTCTGGTGCACAGAGCAAATCCAGCTTGTTGCACGCGATTAACCATCCGAAGAGCCGTCCGAATACGAGCCTGGAGATCGAATTCACGCCGCGTGATAACGAAACCGTTCCGTACGATGAGCTGATTAAAGAACTGACGACGTTGAGCCGCCACATCTTTATGGCCAGCCCGGAAAACGTGATCCTTAGCCCGCCTATCAATGCGCCGATTAAAACCTTTATGTTACCGAAACAAGACATTGTGGGTCTGGACCTGGAAAACCTGTACGCGGTCACCAAAACGGACGGCATTCCGATCACGATTCGTGTTACCAGCAATGGTCTGTACTGCTATTTCACTCATTTGGGCTATATCATTCGTTATCCGGTGAAACGCATCATTGATTCTGAGGTTGTCGTTTTCGGCGAAGCAGTCAAGGACAAGAATTGGACTGTGTACCTGATCAAATTGATTGAACCGGTTAACGCCATCAATGACCGCCTGGAAGAGTCGAAATATGTTGAAAGCAAACTGGTGGATATTTGTGATCGTATCGTGTTCAAGAGCAAGAAATATGAAGGCCCGTTCACCACGACCAGCGAAGTTGTTGACATGCTGAGCACCTATCTGCCGAAACAACCTGAGGGTGTGATTCTGTTTTACTCCAAGGGTCCGAAGAGCAACATTGATTTCAAAATCAAGAAAGAGAATACCATTGATCAGACCGCCAACGTTGTGTTCCGCTATATGTCCAGCGAGCCTATCATTTTCGGTGAGTCGAGCATCTTTGTTGAATACAAAAAGTTTAGCAACGATAAGGGTTTTCCGAAAGAATACGGTTCCGGTAAGATTGTGTTGTACAACGGCGTCAATTATCTGAACAACATCTACTGTCTGGAGTACATCAATACCCATAACGAAGTTGGCATTAAGTCTGTTGTCGTCCCGATCAAATTCATCGCGGAGTTCCTGGTTAACGGTGAGATTCTGAAGCCGCGTATTGATAAAACTATGAAATACATTAACTCCGAAGATTACTACGGTAATCAGCATAACATCATCGTCGAGCACTTGCGTGATCAAAGCATTAAGATCGGTGACATCTTTAACGAAGATAAGCTGAGCGATGTAGGCCACCAGTATGCGAACAATGACAAATTTCGCCTGAATCCGGAAGTCAGCTACTTTACGAATAAGCGCACCCGTGGTCCACTGGGTATCCTGAGCAATTATGTTAAAACCCTGTTGATTTCCATGTACTGCTCCAAAACGTTCCTGGACGACAGCAACAAGCGCAAAGTTCTGGCGATCGACTTCGGTAATGGTGCCGATCTGGAGAAGTACTTTTATGGTGAGATCGCATTGCTGGTTGCTACCGACCCGGATGCAGATGCGATCGCCCGTGGCAACGAGCGTTACAATAAGCTGAATAGCGGTATCAAGACCAAATACTACAAATTCGACTATATTCAAGAGACGATCCGCTCGGACACCTTTGTATCCAGCGTGCGTGAGGTGTTTTACTTCGGTAAATTCAACATCATTGACTGGCAATTCGCCATTCACTATAGCTTTCACCCACGCCACTATGCGACGGTCATGAACAACCTGTCTGAGCTGACCGCGAGCGGCGGTAAAGTTCTGATCACCACGATGGACGGTGACAAGCTGTCTAAACTGACCGACAAAAAGACCTTCATTATTCACAAAAATCTCCCGTCGAGCGAGAATTACATGTCCGTCGAAAAGATTGCGGACGACCGTATTGTTGTCTACAACCCGAGCACTATGTCGACCCCAATGACCGAGTATATCATCAAAAAGAATGACATTGTGCGTGTCTTTAATGAATACGGTTTTGTGCTGGTCGACAACGTCGATTTTGCGACCATCATCGAGAGAAGCAAGAAATTCATTAATGGCGCTTCTACGATGGAAGATCGCCCGAGCACGCGTAACTTCTTTGAGCTGAATCGTGGCGCGATTAAGTGCGAGGGCCTGGACGTCGAGGATCTGCTGTCGTATTACGTGGTTTATGTGTTTAGCAAACGTTAATGA(SEQ ID NO:2)
鸟苷酰转移酶(GT)小亚基DNA:
ATGGACGAAATTGTCAAGAATATCCGTGAAGGTACCCACGTTTTACTGCCATTCTACGAGACGCTGCCGGAACTGAACCTGAGCCTGGGTAAAAGCCCTCTGCCGAGCCTGGAGTATGGTGCGAACTATTTTCTGCAGATTTCCCGTGTAAACGATTTGAACCGCATGCCGACGGACATGCTGAAACTGTTCACCCACGACATCATGCTGCCGGAATCTGATCTGGATAAAGTTTACGAGATCTTGAAAATCAATTCAGTGAAGTACTATGGCCGTAGCACCAAGGCCGATGCGGTGGTCGCAGACCTGAGCGCGCGTAACAAACTGTTTAAACGTGAACGTGACGCAATTAAGAGCAATAACCATCTGACCGAGAACAATTTGTACATCAGCGACTACAAGATGTTGACTTTTGACGTGTTTCGTCCGCTGTTCGACTTTGTTAATGAGAAATACTGCATTATCAAGCTGCCGACGTTGTTTGGTCGCGGCGTCATTGATACGATGCGCATTTACTGCTCTCTCTTCAAGAATGTGCGCCTGCTGAAGTGTGTCTCCGACAGCTGGCTGAAAGATAGCGCTATTATGGTTGCGAGCGACGTGTGTAAAAAGAACCTGGATCTGTTCATGAGCCACGTGAAGAGCGTTACCAAAAGCAGCAGCTGGAAAGACGTTAACAGCGTCCAGTTCTCCATTCTGAATAACCCGGTCGATACCGAGTTTATCAACAAGTTCCTTGAATTCAGCAATCGCGTTTATGAGGCCCTGTATTACGTTCATAGCCTGCTGTATAGCTCCATGACCTCTGATAGCAAATCGATCGAGAATAAACACCAACGTCGTCTGGTGAAACTGCTGCTGTAATGA(SEQ ID NO:3)
具有His标签和接头的SUMO-GT大亚基DNA构建体:
ATGGGCCATCATCATCACCATCACGGCAGCCTGCAAGAAGAGAAACCGAAAGAGGGCGTTAAGACCGAGAATGACCACATTAACCTGAAGGTCGCTGGTCAAGATGGCAGCGTGGTGCAGTTTAAGATCAAGCGTCACACGCCGTTGAGCAAGCTGATGAAGGCTTACTGCGAGCGTCAGGGTCTGAGCATGCGTCAGATCCGCTTTCGTTTCGATGGCCAGCCGATCAATGAGACTGACACCCCAGCGCAACTGGAGATGGAAGATGAAGATACCATCGACGTCTTTCAGCAACAGACCGGTGGTATGGATGCTAACGTCGTTAGCAGCAGCACCATTGCGACTTACATTGATGCACTGGCCAAAAACGCATCTGAGCTTGAGCAGCGCAGCACCGCCTACGAGATCAATAACGAATTGGAGCTGGTTTTCATTAAACCGCCGCTGATCACGCTGACGAACGTCGTGAACATTAGCACGATTCAAGAGAGCTTTATTCGTTTCACCGTTACCAATAAAGAAGGCGTGAAGATCCGTACCAAGATTCCGCTGAGCAAAGTGCATGGTCTGGACGTGAAAAATGTGCAGCTGGTTGATGCGATCGATAACATCGTGTGGGAGAAGAAATCTTTGGTCACGGAAAATCGTCTGCACAAGGAATGTCTGCTGCGTCTGTCAACCGAAGAACGCCACATCTTCCTGGACTACAAGAAGTATGGTTCCAGCATCCGTCTGGAACTGGTGAACCTGATTCAGGCAAAGACCAAGAACTTCACCATTGACTTCAAACTGAAGTATTTCCTGGGCTCTGGTGCACAGAGCAAATCCAGCTTGTTGCACGCGATTAACCATCCGAAGAGCCGTCCGAATACGAGCCTGGAGATCGAATTCACGCCGCGTGATAACGAAACCGTTCCGTACGATGAGCTGATTAAAGAACTGACGACGTTGAGCCGCCACATCTTTATGGCCAGCCCGGAAAACGTGATCCTTAGCCCGCCTATCAATGCGCCGATTAAAACCTTTATGTTACCGAAACAAGACATTGTGGGTCTGGACCTGGAAAACCTGTACGCGGTCACCAAAACGGACGGCATTCCGATCACGATTCGTGTTACCAGCAATGGTCTGTACTGCTATTTCACTCATTTGGGCTATATCATTCGTTATCCGGTGAAACGCATCATTGATTCTGAGGTTGTCGTTTTCGGCGAAGCAGTCAAGGACAAGAATTGGACTGTGTACCTGATCAAATTGATTGAACCGGTTAACGCCATCAATGACCGCCTGGAAGAGTCGAAATATGTTGAAAGCAAACTGGTGGATATTTGTGATCGTATCGTGTTCAAGAGCAAGAAATATGAAGGCCCGTTCACCACGACCAGCGAAGTTGTTGACATGCTGAGCACCTATCTGCCGAAACAACCTGAGGGTGTGATTCTGTTTTACTCCAAGGGTCCGAAGAGCAACATTGATTTCAAAATCAAGAAAGAGAATACCATTGATCAGACCGCCAACGTTGTGTTCCGCTATATGTCCAGCGAGCCTATCATTTTCGGTGAGTCGAGCATCTTTGTTGAATACAAAAAGTTTAGCAACGATAAGGGTTTTCCGAAAGAATACGGTTCCGGTAAGATTGTGTTGTACAACGGCGTCAATTATCTGAACAACATCTACTGTCTGGAGTACATCAATACCCATAACGAAGTTGGCATTAAGTCTGTTGTCGTCCCGATCAAATTCATCGCGGAGTTCCTGGTTAACGGTGAGATTCTGAAGCCGCGTATTGATAAAACTATGAAATACATTAACTCCGAAGATTACTACGGTAATCAGCATAACATCATCGTCGAGCACTTGCGTGATCAAAGCATTAAGATCGGTGACATCTTTAACGAAGATAAGCTGAGCGATGTAGGCCACCAGTATGCGAACAATGACAAATTTCGCCTGAATCCGGAAGTCAGCTACTTTACGAATAAGCGCACCCGTGGTCCACTGGGTATCCTGAGCAATTATGTTAAAACCCTGTTGATTTCCATGTACTGCTCCAAAACGTTCCTGGACGACAGCAACAAGCGCAAAGTTCTGGCGATCGACTTCGGTAATGGTGCCGATCTGGAGAAGTACTTTTATGGTGAGATCGCATTGCTGGTTGCTACCGACCCGGATGCAGATGCGATCGCCCGTGGCAACGAGCGTTACAATAAGCTGAATAGCGGTATCAAGACCAAATACTACAAATTCGACTATATTCAAGAGACGATCCGCTCGGACACCTTTGTATCCAGCGTGCGTGAGGTGTTTTACTTCGGTAAATTCAACATCATTGACTGGCAATTCGCCATTCACTATAGCTTTCACCCACGCCACTATGCGACGGTCATGAACAACCTGTCTGAGCTGACCGCGAGCGGCGGTAAAGTTCTGATCACCACGATGGACGGTGACAAGCTGTCTAAACTGACCGACAAAAAGACCTTCATTATTCACAAAAATCTCCCGTCGAGCGAGAATTACATGTCCGTCGAAAAGATTGCGGACGACCGTATTGTTGTCTACAACCCGAGCACTATGTCGACCCCAATGACCGAGTATATCATCAAAAAGAATGACATTGTGCGTGTCTTTAATGAATACGGTTTTGTGCTGGTCGACAACGTCGATTTTGCGACCATCATCGAGAGAAGCAAGAAATTCATTAATGGCGCTTCTACGATGGAAGATCGCCCGAGCACGCGTAACTTCTTTGAGCTGAATCGTGGCGCGATTAAGTGCGAGGGCCTGGACGTCGAGGATCTGCTGTCGTATTACGTGGTTTATGTGTTTAGCAAACGTTAATGA(SEQ ID NO:4)
小泛素样改性剂(SUMO)蛋白:
EEKPKEGVKTENDHINLKVAGQDGSVVQFKIKRHTPLSKLMKAYCERQGLSMRQIRFRFDGQPINETDTPAQLEMEDEDTIDVFQQQTGG(SEQID NO:1)
鸟苷酰转移酶(GT)大亚基蛋白质:
MDANVVSSSTIATYIDALAKNASELEQRSTAYEINNELELVFIKPPLITLTNVVNISTIQESFIRFTVTNKEGVKIRTKIPLSKVHGLDVKNVQLVDAIDNIVWEKKSLVTENRLHKECLLRLSTEERHIFLDYKKYGSSIRLELVNLIQAKTKNFTIDFKLKYFLGSGAQSKSSLLHAINHPKSRPNTSLEIEFTPRDNETVPYDELIKELTTLSRHIFMASPENVILSPPINAPIKTFMLPKQDIVGLDLENLYAVTKTDGIPITIRVTSNGLYCYFTHLGYIIRYPVKRIIDSEVVVFGEAVKDKNWTVYLIKLIEPVNAINDRLEESKYVESKLVDICDRIVFKSKKYEGPFTTTSEVVDMLSTYLPKQPEGVILFYSKGPKSNIDFKIKKENTIDQTANVVFRYMSSEPIIFGESSIFVEYKKFSNDKGFPKEYGSGKIVLYNGVNYLNNIYCLEYINTHNEVGIKSVVVPIKFIAEFLVNGEILKPRIDKTMKYINSEDYYGNQHNIIVEHLRDQSIKIGDIFNEDKLSDVGHQYANNDKFRLNPEVSYFTNKRTRGPLGILSNYVKTLLISMYCSKTFLDDSNKRKVLAIDFGNGADLEKYFYGEIALLVATDPDADAIARGNERYNKLNSGIKTKYYKFDYIQETIRSDTFVSSVREVFYFGKFNIIDWQFAIHYSFHPRHYATVMNNLSELTASGGKVLITTMDGDKLSKLTDKKTFIIHKNLPSSENYMSVEKIADDRIVVYNPSTMSTPMTEYIIKKNDIVRVFNEYGFVLVDNVDFATIIERSKKFINGASTMEDRPSTRNFFELNRGAIKCEGLDVEDLLSYYVVYVFSKR(SEQ ID NO:6)
鸟苷酰转移酶(GT)小亚基蛋白:
MDEIVKNIREGTHVLLPFYETLPELNLSLGKSPLPSLEYGANYFLQISRVNDLNRMPTDMLKLFTHDIMLPESDLDKVYEILKINSVKYYGRSTKADAVVADLSARNKLFKRERDAIKSNNHLTENNLYISDYKMLTFDVFRPLFDFVNEKYCIIKLPTLFGRGVIDTMRIYCSLFKNVRLLKCVSDSWLKDSAIMVASDVCKKNLDLFMSHVKSVTKSSSWKDVNSVQFSILNNPVDTEFINKFLEFSNRVYEALYYVHSLLYSSMTSDSKSIENKHQRRLVKLLL(SEQ IDNO:7)
具有His标签和接头的SUMO-GT大亚基蛋白:
MGHHHHHHGSLQEEKPKEGVKTENDHINLKVAGQDGSVVQFKIKRHTPLSKLMKAYCERQGLSMRQIRFRFDGQPINETDTPAQLEMEDEDTIDVFQQQTGGMDANVVSSSTIATYIDALAKNASELEQRSTAYEINNELELVFIKPPLITLTNVVNISTIQESFIRFTVTNKEGVKIRTKIPLSKVHGLDVKNVQLVDAIDNIVWEKKSLVTENRLHKECLLRLSTEERHIFLDYKKYGSSIRLELVNLIQAKTKNFTIDFKLKYFLGSGAQSKSSLLHAINHPKSRPNTSLEIEFTPRDNETVPYDELIKELTTLSRHIFMASPENVILSPPINAPIKTFMLPKQDIVGLDLENLYAVTKTDGIPITIRVTSNGLYCYFTHLGYIIRYPVKRIIDSEVVVFGEAVKDKNWTVYLIKLIEPVNAINDRLEESKYVESKLVDICDRIVFKSKKYEGPFTTTSEVVDMLSTYLPKQPEGVILFYSKGPKSNIDFKIKKENTIDQTANVVFRYMSSEPIIFGESSIFVEYKKFSNDKGFPKEYGSGKIVLYNGVNYLNNIYCLEYINTHNEVGIKSVVVPIKFIAEFLVNGEILKPRIDKTMKYINSEDYYGNQHNIIVEHLRDQSIKIGDIFNEDKLSDVGHQYANNDKFRLNPEVSYFTNKRTRGPLGILSNYVKTLLISMYCSKTFLDDSNKRKVLAIDFGNGADLEKYFYGEIALLVATDPDADAIARGNERYNKLNSGIKTKYYKFDYIQETIRSDTFVSSVREVFYFGKFNIIDWQFAIHYSFHPRHYATVMNNLSELTASGGKVLITTMDGDKLSKLTDKKTFIIHKNLPSSENYMSVEKIADDRIVVYNPSTMSTPMTEYIIKKNDIVRVFNEYGFVLVDNVDFATIIERSKKFINGASTMEDRPSTRNFFELNRGAIKCEGLDVEDLLSYYVVYVFSKR(SEQ ID NO:8)
实例2:SUMO-GT蛋白的生产
摇瓶
SUMO-GT融合蛋白的生产可根据标准方法和程序执行。例如,为了测试和比较GT和SUMO-GT融合蛋白的表达,将含有每种SUMO-eGFP质粒的大肠杆菌Rosetta菌株(Novagen)的单个菌落接种到5ml Luria-Bertani(LB)培养基内,所述LB培养基含有100μg/ml卡那霉素和30μg/ml氯霉素。该菌株源自λDE3溶素原菌株,并且携带IPTG诱导型T7 RNA聚合酶的染色体拷贝连同基于pACYC的质粒上的tRNA。细胞在37℃下伴随以250rpm的振荡生长过夜。第二天早上,将过夜培养物转移到100ml新鲜培养基内,以允许指数生长。当OD600值达到-0.6-0.7时,通过加入1mM IPTG(异丙基-β-D-硫代半乳糖吡喃糖苷)诱导蛋白质表达,随后为在37℃下3小时或在20℃下过夜(约15小时)的延长培养。
在通过离心(在4℃下8,000xg共10分钟)从LB培养基(100ml)中收获大肠杆菌细胞后,将细胞团块悬浮于6ml裂解缓冲液(含有300mM NaCl、10mM咪唑、0.1%Triton XlOO和1mM PMSF,pH 8.0的PBS)中。通过超声处理(对于5x30秒脉冲,在50%输出下)裂解细胞。超声处理用在湿冰中加套的管以及在脉冲周期之间的1分钟间隔进行,以防止加热。将裂解产物与DNase和RNase(各自以40μg/ml)一起温育15分钟以消化核酸后,将它们在4℃下以20,000g离心30分钟,并且收集上清液(可溶性蛋白质级分)。将团块用6ml裂解缓冲液洗涤一次,以进一步提取可溶性级分;将洗剂(6ml)与先前的提取物(6ml)组合,以制备12ml的最终体积用于可溶性蛋白质样品。
从大肠杆菌包涵体制备不溶性蛋白质样品。简言之,在去除含有可溶性蛋白质的提取物后,将含有包涵体的团块悬浮于含有50mM CAPS(pH 11.0)、0.3%N-月桂基肌氨酸和1mM DTT的变性溶解缓冲液(Novagen)中,并且在室温下伴随振荡温育20分钟。通过高速离心(在4℃下80,000xg共20分钟)获得提取物(不溶性蛋白质级分)。
为了使用SDS-PAGE检测所表达的蛋白质,将5μl上文制备的样品与3μl含有SDS和β-巯基乙醇的SDSPAGE样品缓冲液混合,并且在95℃下加热5分钟以促进蛋白质的变性和还原。使用具有Tris-甘氨酸运行缓冲液和考马斯蓝染色的15%SDS-聚丙烯酰胺凝胶显现蛋白质。
发酵
最终的SUMO-GT复合酶的溶解度中的大量增加也通过发酵再现。根据标准方法和程序执行发酵。例如,用于生产SUMO-GT融合蛋白的发酵方法包括细胞裂解、固定金属亲和层析(IMAC)、阳离子交换层析、阴离子交换层析和切向流过滤(TFF)形式(formulation)。来源于发酵的SUMO-GT融合蛋白的质量测试包括还原性SDS PAGE以确定纯度和同一性,反相HPLC以确定纯度,浓度的A280测量和鲎变形细胞裂解产物(LAL)测定以测试内毒素。
如图2所示,通过发酵产生的可溶性SUMO-GT蛋白的产率是与经由摇瓶方法产生的GT蛋白的产率可比较的。
等价方案
本领域技术人员将认识到或能够使用不超过常规实验确定本文描述的本发明的具体实施例的许多等价物。本发明的范围并不预期限于上文说明书,而是如权利要求中阐述的。
序列表
<110> 川斯勒佰尔公司
<120> 用于增强生产的RNA相关酶的修饰
<130> SHR-1187WO
<150> 62/241,350
<151> 2015-10-14
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 90
<212> PRT
<213> 人工序列
<220>
<223> 化学合成的多肽
<400> 1
Glu Glu Lys Pro Lys Glu Gly Val Lys Thr Glu Asn Asp His Ile Asn
1 5 10 15
Leu Lys Val Ala Gly Gln Asp Gly Ser Val Val Gln Phe Lys Ile Lys
20 25 30
Arg His Thr Pro Leu Ser Lys Leu Met Lys Ala Tyr Cys Glu Arg Gln
35 40 45
Gly Leu Ser Met Arg Gln Ile Arg Phe Arg Phe Asp Gly Gln Pro Ile
50 55 60
Asn Glu Thr Asp Thr Pro Ala Gln Leu Glu Met Glu Asp Glu Asp Thr
65 70 75 80
Ile Asp Val Phe Gln Gln Gln Thr Gly Gly
85 90
<210> 2
<211> 2588
<212> DNA
<213> 人工序列
<220>
<223> 化学合成的寡核苷酸
<400> 2
agatggaaga tgaagatacc atcgacgtct ttcagcaaca gaccggtggt atggatgcta 60
acgtcgttag cagcagcacc attgcgactt acattgatgc actggccaaa aacgcatctg 120
agcttgagca gcgcagcacc gcctacgaga tcaataacga attggagctg gttttcatta 180
aaccgccgct gatcacgctg acgaacgtcg tgaacattag cacgattcaa gagagcttta 240
ttcgtttcac cgttaccaat aaagaaggcg tgaagatccg taccaagatt ccgctgagca 300
aagtgcatgg tctggacgtg aaaaatgtgc agctggttga tgcgatcgat aacatcgtgt 360
gggagaagaa atctttggtc acggaaaatc gtctgcacaa ggaatgtctg ctgcgtctgt 420
caaccgaaga acgccacatc ttcctggact acaagaagta tggttccagc atccgtctgg 480
aactggtgaa cctgattcag gcaaagacca agaacttcac cattgacttc aaactgaagt 540
atttcctggg ctctggtgca cagagcaaat ccagcttgtt gcacgcgatt aaccatccga 600
agagccgtcc gaatacgagc ctggagatcg aattcacgcc gcgtgataac gaaaccgttc 660
cgtacgatga gctgattaaa gaactgacga cgttgagccg ccacatcttt atggccagcc 720
cggaaaacgt gatccttagc ccgcctatca atgcgccgat taaaaccttt atgttaccga 780
aacaagacat tgtgggtctg gacctggaaa acctgtacgc ggtcaccaaa acggacggca 840
ttccgatcac gattcgtgtt accagcaatg gtctgtactg ctatttcact catttgggct 900
atatcattcg ttatccggtg aaacgcatca ttgattctga ggttgtcgtt ttcggcgaag 960
cagtcaagga caagaattgg actgtgtacc tgatcaaatt gattgaaccg gttaacgcca 1020
tcaatgaccg cctggaagag tcgaaatatg ttgaaagcaa actggtggat atttgtgatc 1080
gtatcgtgtt caagagcaag aaatatgaag gcccgttcac cacgaccagc gaagttgttg 1140
acatgctgag cacctatctg ccgaaacaac ctgagggtgt gattctgttt tactccaagg 1200
gtccgaagag caacattgat ttcaaaatca agaaagagaa taccattgat cagaccgcca 1260
acgttgtgtt ccgctatatg tccagcgagc ctatcatttt cggtgagtcg agcatctttg 1320
ttgaatacaa aaagtttagc aacgataagg gttttccgaa agaatacggt tccggtaaga 1380
ttgtgttgta caacggcgtc aattatctga acaacatcta ctgtctggag tacatcaata 1440
cccataacga agttggcatt aagtctgttg tcgtcccgat caaattcatc gcggagttcc 1500
tggttaacgg tgagattctg aagccgcgta ttgataaaac tatgaaatac attaactccg 1560
aagattacta cggtaatcag cataacatca tcgtcgagca cttgcgtgat caaagcatta 1620
agatcggtga catctttaac gaagataagc tgagcgatgt aggccaccag tatgcgaaca 1680
atgacaaatt tcgcctgaat ccggaagtca gctactttac gaataagcgc acccgtggtc 1740
cactgggtat cctgagcaat tatgttaaaa ccctgttgat ttccatgtac tgctccaaaa 1800
cgttcctgga cgacagcaac aagcgcaaag ttctggcgat cgacttcggt aatggtgccg 1860
atctggagaa gtacttttat ggtgagatcg cattgctggt tgctaccgac ccggatgcag 1920
atgcgatcgc ccgtggcaac gagcgttaca ataagctgaa tagcggtatc aagaccaaat 1980
actacaaatt cgactatatt caagagacga tccgctcgga cacctttgta tccagcgtgc 2040
gtgaggtgtt ttacttcggt aaattcaaca tcattgactg gcaattcgcc attcactata 2100
gctttcaccc acgccactat gcgacggtca tgaacaacct gtctgagctg accgcgagcg 2160
gcggtaaagt tctgatcacc acgatggacg gtgacaagct gtctaaactg accgacaaaa 2220
agaccttcat tattcacaaa aatctcccgt cgagcgagaa ttacatgtcc gtcgaaaaga 2280
ttgcggacga ccgtattgtt gtctacaacc cgagcactat gtcgacccca atgaccgagt 2340
atatcatcaa aaagaatgac attgtgcgtg tctttaatga atacggtttt gtgctggtcg 2400
acaacgtcga ttttgcgacc atcatcgaga gaagcaagaa attcattaat ggcgcttcta 2460
cgatggaaga tcgcccgagc acgcgtaact tctttgagct gaatcgtggc gcgattaagt 2520
gcgagggcct ggacgtcgag gatctgctgt cgtattacgt ggtttatgtg tttagcaaac 2580
gttaatga 2588
<210> 3
<211> 867
<212> DNA
<213> 人工序列
<220>
<223> 化学合成的寡核苷酸
<400> 3
atggacgaaa ttgtcaagaa tatccgtgaa ggtacccacg ttttactgcc attctacgag 60
acgctgccgg aactgaacct gagcctgggt aaaagccctc tgccgagcct ggagtatggt 120
gcgaactatt ttctgcagat ttcccgtgta aacgatttga accgcatgcc gacggacatg 180
ctgaaactgt tcacccacga catcatgctg ccggaatctg atctggataa agtttacgag 240
atcttgaaaa tcaattcagt gaagtactat ggccgtagca ccaaggccga tgcggtggtc 300
gcagacctga gcgcgcgtaa caaactgttt aaacgtgaac gtgacgcaat taagagcaat 360
aaccatctga ccgagaacaa tttgtacatc agcgactaca agatgttgac ttttgacgtg 420
tttcgtccgc tgttcgactt tgttaatgag aaatactgca ttatcaagct gccgacgttg 480
tttggtcgcg gcgtcattga tacgatgcgc atttactgct ctctcttcaa gaatgtgcgc 540
ctgctgaagt gtgtctccga cagctggctg aaagatagcg ctattatggt tgcgagcgac 600
gtgtgtaaaa agaacctgga tctgttcatg agccacgtga agagcgttac caaaagcagc 660
agctggaaag acgttaacag cgtccagttc tccattctga ataacccggt cgataccgag 720
tttatcaaca agttccttga attcagcaat cgcgtttatg aggccctgta ttacgttcat 780
agcctgctgt atagctccat gacctctgat agcaaatcga tcgagaataa acaccaacgt 840
cgtctggtga aactgctgct gtaatga 867
<210> 4
<211> 2844
<212> DNA
<213> 人工序列
<220>
<223> 化学合成的寡核苷酸
<400> 4
atgggccatc atcatcacca tcacggcagc ctgcaagaag agaaaccgaa agagggcgtt 60
aagaccgaga atgaccacat taacctgaag gtcgctggtc aagatggcag cgtggtgcag 120
tttaagatca agcgtcacac gccgttgagc aagctgatga aggcttactg cgagcgtcag 180
ggtctgagca tgcgtcagat ccgctttcgt ttcgatggcc agccgatcaa tgagactgac 240
accccagcgc aactggagat ggaagatgaa gataccatcg acgtctttca gcaacagacc 300
ggtggtatgg atgctaacgt cgttagcagc agcaccattg cgacttacat tgatgcactg 360
gccaaaaacg catctgagct tgagcagcgc agcaccgcct acgagatcaa taacgaattg 420
gagctggttt tcattaaacc gccgctgatc acgctgacga acgtcgtgaa cattagcacg 480
attcaagaga gctttattcg tttcaccgtt accaataaag aaggcgtgaa gatccgtacc 540
aagattccgc tgagcaaagt gcatggtctg gacgtgaaaa atgtgcagct ggttgatgcg 600
atcgataaca tcgtgtggga gaagaaatct ttggtcacgg aaaatcgtct gcacaaggaa 660
tgtctgctgc gtctgtcaac cgaagaacgc cacatcttcc tggactacaa gaagtatggt 720
tccagcatcc gtctggaact ggtgaacctg attcaggcaa agaccaagaa cttcaccatt 780
gacttcaaac tgaagtattt cctgggctct ggtgcacaga gcaaatccag cttgttgcac 840
gcgattaacc atccgaagag ccgtccgaat acgagcctgg agatcgaatt cacgccgcgt 900
gataacgaaa ccgttccgta cgatgagctg attaaagaac tgacgacgtt gagccgccac 960
atctttatgg ccagcccgga aaacgtgatc cttagcccgc ctatcaatgc gccgattaaa 1020
acctttatgt taccgaaaca agacattgtg ggtctggacc tggaaaacct gtacgcggtc 1080
accaaaacgg acggcattcc gatcacgatt cgtgttacca gcaatggtct gtactgctat 1140
ttcactcatt tgggctatat cattcgttat ccggtgaaac gcatcattga ttctgaggtt 1200
gtcgttttcg gcgaagcagt caaggacaag aattggactg tgtacctgat caaattgatt 1260
gaaccggtta acgccatcaa tgaccgcctg gaagagtcga aatatgttga aagcaaactg 1320
gtggatattt gtgatcgtat cgtgttcaag agcaagaaat atgaaggccc gttcaccacg 1380
accagcgaag ttgttgacat gctgagcacc tatctgccga aacaacctga gggtgtgatt 1440
ctgttttact ccaagggtcc gaagagcaac attgatttca aaatcaagaa agagaatacc 1500
attgatcaga ccgccaacgt tgtgttccgc tatatgtcca gcgagcctat cattttcggt 1560
gagtcgagca tctttgttga atacaaaaag tttagcaacg ataagggttt tccgaaagaa 1620
tacggttccg gtaagattgt gttgtacaac ggcgtcaatt atctgaacaa catctactgt 1680
ctggagtaca tcaataccca taacgaagtt ggcattaagt ctgttgtcgt cccgatcaaa 1740
ttcatcgcgg agttcctggt taacggtgag attctgaagc cgcgtattga taaaactatg 1800
aaatacatta actccgaaga ttactacggt aatcagcata acatcatcgt cgagcacttg 1860
cgtgatcaaa gcattaagat cggtgacatc tttaacgaag ataagctgag cgatgtaggc 1920
caccagtatg cgaacaatga caaatttcgc ctgaatccgg aagtcagcta ctttacgaat 1980
aagcgcaccc gtggtccact gggtatcctg agcaattatg ttaaaaccct gttgatttcc 2040
atgtactgct ccaaaacgtt cctggacgac agcaacaagc gcaaagttct ggcgatcgac 2100
ttcggtaatg gtgccgatct ggagaagtac ttttatggtg agatcgcatt gctggttgct 2160
accgacccgg atgcagatgc gatcgcccgt ggcaacgagc gttacaataa gctgaatagc 2220
ggtatcaaga ccaaatacta caaattcgac tatattcaag agacgatccg ctcggacacc 2280
tttgtatcca gcgtgcgtga ggtgttttac ttcggtaaat tcaacatcat tgactggcaa 2340
ttcgccattc actatagctt tcacccacgc cactatgcga cggtcatgaa caacctgtct 2400
gagctgaccg cgagcggcgg taaagttctg atcaccacga tggacggtga caagctgtct 2460
aaactgaccg acaaaaagac cttcattatt cacaaaaatc tcccgtcgag cgagaattac 2520
atgtccgtcg aaaagattgc ggacgaccgt attgttgtct acaacccgag cactatgtcg 2580
accccaatga ccgagtatat catcaaaaag aatgacattg tgcgtgtctt taatgaatac 2640
ggttttgtgc tggtcgacaa cgtcgatttt gcgaccatca tcgagagaag caagaaattc 2700
attaatggcg cttctacgat ggaagatcgc ccgagcacgc gtaacttctt tgagctgaat 2760
cgtggcgcga ttaagtgcga gggcctggac gtcgaggatc tgctgtcgta ttacgtggtt 2820
tatgtgttta gcaaacgtta atga 2844
<210> 5
<211> 220
<212> DNA
<213> 人工序列
<220>
<223> 化学合成的寡核苷酸
<400> 5
gaagagaaac cgaaagaggg cgttaagacc gagaatgacc acattaacct gaaggtcgct 60
ggtcaagatg gcagcgtggt gcagtttaag atcaagcgtc acacgccgtt gagcaagctg 120
atgaaggctt actgcgagcg tcagggtctg agcatgcgtc agatccgctt tcgtttcgat 180
ggccagccga tcaatgagac tgacacccca gcgcaactgg 220
<210> 6
<211> 844
<212> PRT
<213> 人工序列
<220>
<223> 化学合成的多肽
<400> 6
Met Asp Ala Asn Val Val Ser Ser Ser Thr Ile Ala Thr Tyr Ile Asp
1 5 10 15
Ala Leu Ala Lys Asn Ala Ser Glu Leu Glu Gln Arg Ser Thr Ala Tyr
20 25 30
Glu Ile Asn Asn Glu Leu Glu Leu Val Phe Ile Lys Pro Pro Leu Ile
35 40 45
Thr Leu Thr Asn Val Val Asn Ile Ser Thr Ile Gln Glu Ser Phe Ile
50 55 60
Arg Phe Thr Val Thr Asn Lys Glu Gly Val Lys Ile Arg Thr Lys Ile
65 70 75 80
Pro Leu Ser Lys Val His Gly Leu Asp Val Lys Asn Val Gln Leu Val
85 90 95
Asp Ala Ile Asp Asn Ile Val Trp Glu Lys Lys Ser Leu Val Thr Glu
100 105 110
Asn Arg Leu His Lys Glu Cys Leu Leu Arg Leu Ser Thr Glu Glu Arg
115 120 125
His Ile Phe Leu Asp Tyr Lys Lys Tyr Gly Ser Ser Ile Arg Leu Glu
130 135 140
Leu Val Asn Leu Ile Gln Ala Lys Thr Lys Asn Phe Thr Ile Asp Phe
145 150 155 160
Lys Leu Lys Tyr Phe Leu Gly Ser Gly Ala Gln Ser Lys Ser Ser Leu
165 170 175
Leu His Ala Ile Asn His Pro Lys Ser Arg Pro Asn Thr Ser Leu Glu
180 185 190
Ile Glu Phe Thr Pro Arg Asp Asn Glu Thr Val Pro Tyr Asp Glu Leu
195 200 205
Ile Lys Glu Leu Thr Thr Leu Ser Arg His Ile Phe Met Ala Ser Pro
210 215 220
Glu Asn Val Ile Leu Ser Pro Pro Ile Asn Ala Pro Ile Lys Thr Phe
225 230 235 240
Met Leu Pro Lys Gln Asp Ile Val Gly Leu Asp Leu Glu Asn Leu Tyr
245 250 255
Ala Val Thr Lys Thr Asp Gly Ile Pro Ile Thr Ile Arg Val Thr Ser
260 265 270
Asn Gly Leu Tyr Cys Tyr Phe Thr His Leu Gly Tyr Ile Ile Arg Tyr
275 280 285
Pro Val Lys Arg Ile Ile Asp Ser Glu Val Val Val Phe Gly Glu Ala
290 295 300
Val Lys Asp Lys Asn Trp Thr Val Tyr Leu Ile Lys Leu Ile Glu Pro
305 310 315 320
Val Asn Ala Ile Asn Asp Arg Leu Glu Glu Ser Lys Tyr Val Glu Ser
325 330 335
Lys Leu Val Asp Ile Cys Asp Arg Ile Val Phe Lys Ser Lys Lys Tyr
340 345 350
Glu Gly Pro Phe Thr Thr Thr Ser Glu Val Val Asp Met Leu Ser Thr
355 360 365
Tyr Leu Pro Lys Gln Pro Glu Gly Val Ile Leu Phe Tyr Ser Lys Gly
370 375 380
Pro Lys Ser Asn Ile Asp Phe Lys Ile Lys Lys Glu Asn Thr Ile Asp
385 390 395 400
Gln Thr Ala Asn Val Val Phe Arg Tyr Met Ser Ser Glu Pro Ile Ile
405 410 415
Phe Gly Glu Ser Ser Ile Phe Val Glu Tyr Lys Lys Phe Ser Asn Asp
420 425 430
Lys Gly Phe Pro Lys Glu Tyr Gly Ser Gly Lys Ile Val Leu Tyr Asn
435 440 445
Gly Val Asn Tyr Leu Asn Asn Ile Tyr Cys Leu Glu Tyr Ile Asn Thr
450 455 460
His Asn Glu Val Gly Ile Lys Ser Val Val Val Pro Ile Lys Phe Ile
465 470 475 480
Ala Glu Phe Leu Val Asn Gly Glu Ile Leu Lys Pro Arg Ile Asp Lys
485 490 495
Thr Met Lys Tyr Ile Asn Ser Glu Asp Tyr Tyr Gly Asn Gln His Asn
500 505 510
Ile Ile Val Glu His Leu Arg Asp Gln Ser Ile Lys Ile Gly Asp Ile
515 520 525
Phe Asn Glu Asp Lys Leu Ser Asp Val Gly His Gln Tyr Ala Asn Asn
530 535 540
Asp Lys Phe Arg Leu Asn Pro Glu Val Ser Tyr Phe Thr Asn Lys Arg
545 550 555 560
Thr Arg Gly Pro Leu Gly Ile Leu Ser Asn Tyr Val Lys Thr Leu Leu
565 570 575
Ile Ser Met Tyr Cys Ser Lys Thr Phe Leu Asp Asp Ser Asn Lys Arg
580 585 590
Lys Val Leu Ala Ile Asp Phe Gly Asn Gly Ala Asp Leu Glu Lys Tyr
595 600 605
Phe Tyr Gly Glu Ile Ala Leu Leu Val Ala Thr Asp Pro Asp Ala Asp
610 615 620
Ala Ile Ala Arg Gly Asn Glu Arg Tyr Asn Lys Leu Asn Ser Gly Ile
625 630 635 640
Lys Thr Lys Tyr Tyr Lys Phe Asp Tyr Ile Gln Glu Thr Ile Arg Ser
645 650 655
Asp Thr Phe Val Ser Ser Val Arg Glu Val Phe Tyr Phe Gly Lys Phe
660 665 670
Asn Ile Ile Asp Trp Gln Phe Ala Ile His Tyr Ser Phe His Pro Arg
675 680 685
His Tyr Ala Thr Val Met Asn Asn Leu Ser Glu Leu Thr Ala Ser Gly
690 695 700
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Claims (29)
1.一种产生加帽的RNA或RNA类似物寡核苷酸的方法,其中融合蛋白促进将鸟苷酰分子转移至所述RNA或RNA类似物寡核苷酸的5’末端和使所述鸟苷酰分子甲基化的步骤。
2.根据权利要求1所述的方法,其中所述融合蛋白包含鸟苷酰转移酶和小泛素样分子(SUMO)蛋白。
3.根据权利要求2所述的方法,其中所述鸟苷酰转移酶包含SEQ ID NO:6和SEQ ID NO:7,并且所述SUMO蛋白包含SEQ ID NO:5。
4.根据权利要求2所述的方法,其中所述融合蛋白包含SEQ ID NO:
8和SEQ ID NO:7。
5.根据权利要求1所述的方法,其中相对于野生型鸟苷酰转移酶蛋白,所述融合蛋白具有可比较的磷酸酶活性、鸟苷酰转移酶活性和甲基化活性。
6.一种融合蛋白,其中所述融合蛋白包含鸟苷酰转移酶和小泛素样分子(SUMO)蛋白。
7.根据权利要求6所述的融合蛋白,其中所述鸟苷酰转移酶包含SEQ ID NO:6和SEQ IDNO:7,并且所述SUMO蛋白包含SEQ ID NO:5。
8.根据权利要求6所述的融合蛋白,其中所述鸟苷酰转移酶包含大亚基和小亚基。
9.根据权利要求8所述的融合蛋白,其中所述SUMO蛋白与所述大亚基共价连接且共表达。
10.根据权利要求6所述的融合蛋白,其中相对于野生型鸟苷酰转移酶蛋白,所述融合蛋白具有可比较的磷酸酶活性、鸟苷酰转移酶活性和甲基化活性。
11.一种载体,所述载体编码包含鸟苷酰转移酶蛋白和小泛素样分子(SUMO)蛋白的融合蛋白。
12.根据权利要求11所述的载体,其中所述载体包含SEQ ID NO:1和SEQ ID NO:2。
13.根据权利要求11所述的载体,其中所述载体包含SEQ ID NO:1、SEQ ID NO:2和SEQID NO:3。
14.根据权利要求11所述的载体,其中所述载体包含SEQ ID NO:4和SEQ ID NO:3。
15.一种通过发酵生产鸟苷酰转移酶的方法,所述方法包括:a)在发酵培养基中培养用至少一种重组核酸分子转化的微生物,所述重组核酸分子包含编码鸟苷酰转移酶的核酸序列,所述鸟苷酰转移酶具有与SEQ ID NO:6和SEQ ID NO:7至少90%相同的氨基酸序列;和b)收集由所述培养步骤产生的产物。
16.根据权利要求15所述的方法,其中所述鸟苷酰转移酶包含鸟苷酰转移酶融合蛋白。
17.根据权利要求16所述的方法,其中相对于野生型鸟苷酰转移酶蛋白,所述鸟苷酰转移酶融合蛋白具有可比较的磷酸酶活性、鸟苷酰转移酶活性和甲基化活性。
18.根据权利要求16所述的方法,其中所述鸟苷酰转移酶融合蛋白包含小泛素样分子(SUMO)蛋白。
19.根据权利要求18所述的方法,其中所述鸟苷酰转移酶融合蛋白包含SEQ ID NO:8。
20.根据权利要求18所述的方法,其中所述SUMO蛋白通过共价键与所述鸟苷酰转移酶结合。
21.根据权利要求20所述的方法,其中所述共价键在所述SUMO蛋白和所述鸟苷酰转移酶的大亚基之间。
22.根据权利要求15所述的方法,其中所述发酵培养基选自Terrific Broth、Cinnabar、2xYT和LB。
23.根据权利要求15所述的方法,其中所述微生物是细菌。
24.根据权利要求15所述的方法,其中编码所述鸟苷酰转移酶的所述核酸序列与SEQID NO:2和SEQ ID NO:3至少90%相同。
25.根据权利要求15的方法,其中所述重组核酸分子还包含编码小泛素样分子(SUMO)蛋白的核酸序列。
26.根据权利要求25所述的方法,其中编码小泛素样分子(SUMO)蛋白的所述核酸序列与SEQ ID NO:1至少90%相同。
27.根据权利要求15所述的方法,其中所述产物是鸟苷酰转移酶。
28.根据权利要求27所述的方法,其中所述产物是包含鸟苷酰转移酶融合蛋白的鸟苷酰转移酶。
29.根据权利要求28所述的方法,其中所述鸟苷酰转移酶融合蛋白还包含小泛素样分子(SUMO)蛋白。
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CN108473969B (zh) | 2022-09-13 |
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CN108473969A (zh) | 2018-08-31 |
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WO2017066573A1 (en) | 2017-04-20 |
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AU2016338559B2 (en) | 2022-11-24 |
EP3878955A1 (en) | 2021-09-15 |
CN108473969B9 (zh) | 2024-08-23 |
MA56219A (fr) | 2022-04-20 |
US20220010347A1 (en) | 2022-01-13 |
US20240175065A1 (en) | 2024-05-30 |
CA3001852A1 (en) | 2017-04-20 |
EP3362555A1 (en) | 2018-08-22 |
US10144942B2 (en) | 2018-12-04 |
AU2016338559A1 (en) | 2018-05-10 |
JP7119181B6 (ja) | 2022-11-11 |
US10995354B2 (en) | 2021-05-04 |
JP6997704B2 (ja) | 2022-02-04 |
US20190136283A1 (en) | 2019-05-09 |
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