CN110938151B - 用于表达甲状旁腺素pth的融合蛋白及重组质粒、重组工程菌 - Google Patents
用于表达甲状旁腺素pth的融合蛋白及重组质粒、重组工程菌 Download PDFInfo
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- CN110938151B CN110938151B CN201911390866.7A CN201911390866A CN110938151B CN 110938151 B CN110938151 B CN 110938151B CN 201911390866 A CN201911390866 A CN 201911390866A CN 110938151 B CN110938151 B CN 110938151B
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Abstract
本申请公开了一种用于表达重组甲状旁腺素PTH的融合蛋白,所述融合蛋白依次由表达增强肽、连接肽1、His6、连接肽2、引导蛋白、连接肽3、蛋白酶切位点和甲状旁腺素PTH(1‑34)组成;本发明还提供了用于表达重组甲状旁腺素PTH的融合蛋白的重组质粒及工程菌。通过本发明可以获得了以近似包涵体表达的融合蛋白及稳定的重组工程菌株。通过对表达蛋白进行蛋白酶酶切及少数步骤的纯化,可以获得纯度高于99.5%、具有良好生物学活性的目的蛋白PTH(1‑34)。
Description
技术领域
本发明属于生物技术制药领域,涉及甲状旁腺素PTH(1-34)的融合蛋白及表达载体。
背景技术
甲状旁腺素由甲状旁腺主细胞合成,含84个氨基酸。甲状旁腺素分泌入血以后,以三种形式存在。第1种,为完整的84个氨基酸(PTH(1-84));第2种,含第1-34位氨基酸(PTH(1-34));第3种,含第55-84位氨基酸。PTH(1-84)和PTH(1-34)均有通过骨骼和肾脏等靶器官调节机体血钙浓度的完全生物活性。PTH(1-34)包含了表现PTH全部生物活性的所有必需结构,具有完整PTH的生物学活性;而在促进成骨细胞生长、刺激骨形成方面更优于PTH(1-84)。
PTH(1-34)的制备,目前有化学合成及生物合成两种手段。化学合成相对容易,但要用到大量有机溶剂;同时PTH(1-34)在合成过程中需对关键氨基酸进行保护与去保护工作,最终成本并不低。因而,用重组大肠杆菌进行PTH(1-34)的生物合成是更优的解决方案。
在重组工程大肠杆菌表达中,常用引导蛋白帮助目的蛋白折叠、提高目的蛋白产量、可溶性及易于纯化等。PTH(1-34)以小分子多肽,单独表达难于纯化,一般都以融合蛋白表达的形式来进行。在表达过程中,融合蛋白以可溶形式表达并不利于PTH(1-34),因其容易被氧化,通过后续纯化得到的被氧化的目的蛋白PTH(1-34)生活学活性大幅降低或丧失;而融合蛋白以形成致密包涵体形式表达,后续包涵体复性及目的蛋白纯化都很困难,尤其可能在PTH(1-34)纯化过程中用到反向层析纯化而使用大量有机溶剂,这将导致经济成本和环境成本的大幅上升。因此,寻找一种使PTH(1-34)在表达过程中不被氧化,在分离纯化过程中得率高、活性好且成本低的融合蛋白表达方法是制备PTH(1-34)的理想方案。
PTH(1-34)氨基端的完整性要求很高,添加、替换、删除原有氨基酸都不能保证其正常生物学活性,因此对融合蛋白中酶切位点的要求高。因子Xa识别位点Ile-Glu/Asp-Gly-Arg↓,肠激酶识别Asp-Asp-Asp-Asp-Lys↓,两者切割融合蛋白后不会在目的蛋白的氨基端留下多余氨基酸,但这两种酶都价格昂贵,也不容易自行制备。
发明内容
针对上述技术问题,发明人发现基于烟草花叶蚀刻病毒蛋白酶(TEV酶)识别一个七个氨基酸的序列Glu-Asn-Leu-Tyr-Phe-Gln↓Gly/Ser,切割发生在Gln和Gly/Ser之间,这样会使得目标蛋白的氨基端多一个Gly或Ser。而PTH(1-34)氨基端第一个氨基酸为丝氨酸(Ser),因此,发明人决定用TEV蛋白酶酶切包含PTH(1-34)的融合蛋白是一个不错的选择。同时TEV酶可以用大肠杆菌表达,制备成本比肠激酶低。
本发明的一方面提供了一种用于表达重组甲状旁腺素PTH的融合蛋白,所述融合蛋白依次由表达增强肽、连接肽1、His6、连接肽2、引导蛋白、连接肽3、蛋白酶切位点和甲状旁腺素PTH(1-34)组成;其中,
所述表达增强肽由信号肽与促表达标签蛋白的组成,所述信号肽选自TorA的N端短肽SEQ ID No.1(MNNNDLFQ)、MdoD的N端短肽SEQ ID No.2(MDRRRFIKGS)、Sufl的N端短肽SEQ ID No.3(MSLSRRQFIQ)、pelB的N端短肽SEQ ID No.4(MKYLLPT)、AlyQ的N端短肽SEQ IDNo.5(MNHKVHM)中的一种;所述促表达标签蛋白选自Myc:SEQ ID No.6(EQKLISEEDL)、HA:SEQ ID No.7(YPYDVPDYA)或Strep tag:SEQ ID No.8(NWSHPQFEK);中的一种;所述信号肽优选为AlyQ的N端短肽SEQ ID No.5(MNHKVHM);所述促表达标签蛋白优选为Strep tag:SEQID No.8(NWSHPQFEK)。
所述连接肽1为具有SSG或其重复结构的肽段;
所述连接肽2为具有GGS或其重复结构的肽段;
所述的引导蛋白,选自谷胱甘肽巯基转移酶GST、类泛素蛋白修饰分子SUMO、麦芽糖结合蛋白MBP、硫氧还蛋白TRX、二硫键形成蛋白DSB或肝片吸虫8-kDa抗原FH8中的一种;优选为SUMO。
所述连接肽3为(GGGGS)n,n=0-4;优选地n=2;
所述蛋白酶切位点选自肠激酶位点,凝血酶位点及烟草蚀刻病毒蛋白酶位点中的一种;优选为烟草蚀刻病毒蛋白酶位点。
在根据本发明的一个实施方案中,所述表达增强肽中信号肽为SEQ ID No.5;促表达标签蛋白为SEQ ID No.8。
在根据本发明的一个实施方案中,所述连接肽1为SSGSSG。
在根据本发明的一个实施方案中,所述连接肽2为为GGSGGS。
在根据本发明的一个实施方案中,所述甲状旁腺素PTH(1-34)的氨基酸序列为SEQID NO:9。
在根据本发明的一个实施方案中,所述融合蛋白的氨基酸序列为SEQ ID NO:12。
本发明的另一方面还提供了一种用于表达重组甲状旁腺素PTH的重组质粒,其包含骨架质粒和表达上述的融合蛋白的核苷酸序列。
在根据本发明的一个实施方案中,所述骨架质粒选自pET系列载体、pGEX系列载体、pMAL系列及pCOLD-SUMO载体中的一种。优选为pET28a。
在根据本发明的一个实施方案中,所述编码融合蛋白的核苷酸序列中编码甲状旁腺素PTH(1-34)的核苷酸序列为SEQ ID NO:10。
本发明的再一个方面提供了一种用于表达甲状旁腺素PTH的重组工程菌,所述重组工程菌含有上述的重组质粒,所述宿主菌为BL21(DE3)。
PTH(1-34)生物合成表达过程中,面临不易表达、易氧化及产量低等许多困难。
本发明亦遇到如下困难:
1)含PTH(1-34)的融合蛋白以包涵体形式表达,包涵体结构紧密,融合蛋白需经过完全变性、完全复性后再进行酶切,这种情况步骤多,尤其纯化目的蛋白PTH(1-34)过程用到反相层析,用到大量有机溶剂,成本高,环境不友好。
2)含PTH(1-34)的融合蛋白能以可溶形式表达,纯化过程得到大幅度简化,但得到的目的蛋白PTH(1-34)主要为其氧化物,生物学活性大幅丧失。
因此本发明通过构建不同的引导蛋白-连接肽-蛋白酶切位点-PTH(1-34)结构单元,重组入表达载体并转化不同大肠杆菌以构建不同的重组工程菌。在初步筛选的基因上,通过调节引导蛋白前面各元件,进行多种形式的组合,反复筛选,充分利用SUMO帮助融合蛋白折叠、增加可溶性的特点,又利用pET28a载体表达量高、表达增强肽进一步提升表达量的特性,最终获得了以近似包涵体表达的融合蛋白及稳定的重组工程菌株。通过对表达蛋白进行蛋白酶酶切及少数步骤的纯化,可以获得纯度高于99.5%、具有良好生物学活性的目的蛋白PTH(1-34)。
本发明的重要意义在于可以帮助制药企业快速、简便且廉价地获得PTH(1-34),解决生产上的难题,增加经济效益。
本发明的特色和创新之处在于:
独特的重组载体构建保证了重组工程菌能在37度培养、0.5mmol/L的IPTG诱导并继续在37度培养的常规情况下,以近似包涵体形式高效表达,这使得目的蛋白在表达过程得到很好保护,不至于被氧化;同时融合蛋白变性后能有效地结合于特异亲和层析填料,并且能在填料上实现复性。
利用对酶切缓冲条件要求宽容的TEV蛋白酶对融合蛋白进行酶切,很好地利用了其切割特异性,保证目的蛋白PTH(1-34)不增减任何氨基酸。
结合于填料上的融合蛋白能被同样可结合于填料的TEV蛋白酶酶切,目的蛋白直接以较高纯度的流穿液形式被收集,简化了后续纯化步骤。
通过对本发明构建的重组工程菌株发酵,其融合蛋白产量高,整个目的蛋白纯化过程步骤少、不涉及常用的反相层析纯化,环境友好;目的蛋白PTH(1-34)纯度达到99.5%及以上,具有与WHO标准品相同生物学活性,其产量超过25mg/L培养基。
附图说明
图1为融合蛋白的包涵体表达情况示意图;其中,M:中分子量蛋白标准;1:全菌蛋白;2:破菌上清;3:破菌沉淀;4:包涵体洗涤离心上清;5:包涵体洗涤离心沉淀经8mol/L尿素溶解;箭头所示为融合蛋白;
图2为以可溶及包涵体形式表达的携带不同酶切位点的融合蛋白的示意图;其中,M:中分子量蛋白标准;1:含TEV蛋白酶切位点的融合蛋白重组工程菌诱导前,2:含肠激酶酶切位点的融合蛋白重组工程菌诱导前;3:含Xa因子酶切位点的融合蛋白重组工程菌诱导前;4:含TEV蛋白酶切位点的融合蛋白重组工程菌诱导后全菌;5:含肠激酶酶切位点的融合蛋白重组工程菌诱导后全菌;6:含Xa因子酶切位点的融合蛋白重组工程菌诱导后全菌;7:含TEV蛋白酶切位点的融合蛋白重组工程菌破菌上清;8:含肠激酶酶切位点的融合蛋白重组工程菌破菌上清;9:含Xa因子酶切位点的融合蛋白重组工程菌破菌上清;10:含TEV蛋白酶切位点的融合蛋白重组工程菌破菌沉淀;11:含肠激酶酶切位点的融合蛋白重组工程菌破菌沉淀;12:含Xa因子酶切位点的融合蛋白重组工程菌破菌沉淀;箭头所示为融合蛋白。
图3为PTH融合蛋白(pET28a-FH8-(GGGGS)2-TEVs-PTH)的分泌表达及纯化结果图;其中,M:中分子量蛋白标准;1:全菌;2:破菌离心沉淀;3:破菌离心上清;4:Ni-NTA亲和流穿;5:酶切前填料;6:酶切后填料;7:酶切后流穿;8:SP纯化1#峰;9:SP纯化2#峰;空心箭头所示为融合蛋白FH8-连接肽-PTH(1-34),实心箭头所示为目的蛋白PTH(1-34);由图可见,融合蛋白主要表达在上清,说明为可溶蛋白;目的蛋白PTH(1-34)在SP纯化2#峰里;
图4为WHO标准品的HPLC结果图;其中,主峰保留时间:21.004min;
图5为包涵体表达PTH(1-34)纯化终产品的HPLC结果图;其中,主峰保留时间:21.079min;
图6为包涵体表达PTH(1-34)纯化终产品H2O2氧化后的HPLC;由图看出,PTH(1-34)依然保留,但大幅减少,同时分别在20.254,20.581及20.733分时出现了三个氧化峰;
图7为分泌表达PTH(1-34)纯化终产品的HPLC结果图;其中,最大峰(91.8%)保留时间:20.719min,该峰实为氧化峰,未氧化的PTH(1-34)仅占3.47%;
图8为包涵体表达PTH(1-34)纯化终产品H2O2氧化与分泌表达PTH(1-34)精纯品的HPLC比较;由图可见,可溶表达的PTH(1-34)经最终纯化后得到的主要为PTH(1-34)的氧化峰。因此,PTH(1-34)不宜通过可溶形式表达;
图9为融合蛋白(表达增强肽-连接肽1-His6-连接肽2-引导蛋白-连接肽3-蛋白酶切位点-PTH(1-34))重组工程菌的60L发酵表达;其中,M:中分子蛋白质标准;1:重组工程菌诱导前;2:重组工程菌经0.5mmol/L IPTG诱导1小时;3:重组工程菌经0.5mmol/L IPTG诱导2小时;4:重组工程菌经0.5mmol/L IPTG诱导3小时;5:重组工程菌经0.5mmol/L IPTG诱导4小时;6:重组工程菌培养上清;7:重组工程菌破菌离心沉淀;8:重组工程菌破菌离心上清;箭头所示为融合蛋白;
图10为发酵表达的融合蛋白在不同条件下经TEV蛋白酶酶切的结果;M:中分子量蛋白标准;1:37度,基础缓冲液,100mmol/L咪唑,TEV酶;2:4度,基础缓冲液,100mmol/L咪唑,TEV酶;3:基础缓冲液,100mmol/L咪唑;4:37度,基础缓冲液,300mmol/L咪唑,TEV酶;5:4度,基础缓冲液,300mmol/L咪唑,TEV酶;6:基础缓冲液,300mmol/L咪唑;7:TEV酶;基础缓冲液:20mmol/L PB,100mmol/L氯化钠,2mol/L尿素,pH 7.1;箭头所示为目的蛋白PTH(1-34);结果表明,融合蛋白能在比较宽松的条件被TEV酶切割;
图11为PTH(1-34)与礼来特立帕肽刺激UMR-106细胞产生cAMP能力的性比较图;如图中所示,PTH(1-34)的生物学活性不低于对照品礼来特立帕肽;
图12为WHO标准品与PTH(1-34)圆二色谱图;图形及数据显示,本申请研制的PTH(1-34)(20190627批)与WHO标准品的二级结构一致;
图13为WHO标准品与PTH(1-34)(20190410批)肽图比较图;由图看出,PTH(1-34)(20190410批)与WHO标准品完全一致。
具体实施方式
以下实施例用于说明本申请,但不用来限制本申请的范围。
下面将更详细地描述本申请的具体实施例。提供这些实施例是为了能够更透彻地理解本申请,并且能够将本申请的范围完整的传达给本领域的技术人员。
如在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语,故应解释成“包含但不限定于”。说明书后续描述为实施本申请的较佳实施方式,然所述描述乃以说明书的一般原则为目的,并非用以限定本申请的范围。本申请的保护范围当视所附权利要求所界定者为准。
实验试剂与材料
1.菌株、质粒
菌株BL21(DE3),DH5alpha大肠杆菌购于Novagen公司;菌株XL-1blue大肠杆菌为美国安捷伦公司产品;质粒pGEX-6p-2为美国GE Healthcare公司产品;质粒pET22b,pET28a,pET32a,pET39b(+)为美国Merck公司产品;pMal-c4X为NEB公司产品;pCOLD-SUMO购自索来宝公司。
2.试剂
WHO PTH(1-34)标准品(英国NIBSC15/304)购自北京市华信行生物科技有限公司,Lilly特立帕肽购自重庆和平欣特健康管理有限公司,UMR-106(大鼠骨肉瘤细胞)-购自ATCC,cAMP Parameter SixPak(6Plates)(R&D公司,美国)购自重庆金麦生物技术有限公司。
TEV酶、EK酶购自碧云天生物技术有限公司,Xa因子蛋白酶北京百奥莱博科技有限公司。大规模酶切融合蛋白所用TEV酶为本公司表达纯化所获得产品。
primeSTAR HS DNA聚合酶、DNA分子量标准、限制性内切酶、DNA连接酶、点突变试剂盒(MutanBest)等为大连宝生物(TakaRa)公司产品;同源重组试剂盒Mut ExpressMultiS Fast Mutagenesis Kit V2购自南京诺唯赞生物科技有限公司;质粒提取试剂盒和凝胶回收试剂盒为美国Omega公司产品;蛋白胨和酵母提取物购自Oxfoid公司,培养基购自北京奥博星生物技术有限责任公司。氨苄西林、卡那霉素为华北制药产品。PBS粉末为中彬金桥产品。蛋白分子量标准为Thermo fisher(美国)公司产品。
HEPES、DMEM细胞培养基、胎牛血清为Gibco(美国)公司产品。细胞培养耗材为Corning(美国)公司产品。
Ni-NTA购自Invitrogen公司。
其余试剂为市售的分析纯产品。
实施例1重组PTH(1-34)工程菌的构建及表达
PTH(1-34)为SEQ ID NO:9的氨基酸序列,将编码DNA密码优化后为SEQ ID NO:10。
设计在PTH(1-34)的N端分别加入TEV酶酶切位点(ENLYFQ)、肠激酶酶位点(DDDDK)、Xa因子酶切位点(IEGR)的三段基因,各段基因从右至左再顺序加入(GGGGS)3、His6及SSGSSG,在各段基因的5’端加入BamHI位点GGATCC,3’端加入终止密码TGATAA、HindIII位点AAGCTT、NotI位点GCGGCCGC,序列交上海生物工程有限公司合成。
分别用BamHI+HindIII及BamHI+NotI双酶切合成的三段基因及如表一中的各载体,分别回收目的片段及载体。按表1的方式进行重组表达载体的构建。
表1重组PTH(1-34)表达载体的构建
交上海生物工程有限公司合成全合成FH8基因(SEQ ID NO:11)并装入pET22b及pET28a,设计引物对扩增编码-(GGGGS)n-TEVs-PTH的DNA序列,设计引物对pET22b、pET28a分别扩增,利用同源重组试剂盒Mut Express MultiS Fast Mutagenesis Kit V2按厂家的说明书进行重组载体pET22b-FH8-(GGGGS)n-TEVs-PTH及pET28a-FH8-(GGGGS)n-TEVs-PTH的构建。重组质粒转化大肠杆菌DH5alpha。
对上述两步骤获得的阳性克隆进行培养、质粒提取,质粒鉴定正确后分别转化大肠杆菌XL1-Blue(pGEX-6P-2)及BL21(DE3)(其余各质粒)。
挑取重组工程菌单菌落进行2L摇瓶表达研究,收获的诱导后细菌用PBS分散并超声破菌,分别用10%SDS-PAGE比较不同工程菌对融合蛋白的表达情况,部分结果见图1、2、3,结果表明有的融合蛋白可溶表达,有的以包涵体形式表达,有的两种情况均有。由图3可见,融合蛋白主要表达在上清,说明为可溶蛋白;目的蛋白PTH(1-34)在SP纯化2#峰里。
实施例2 PTH(1-34)性质初步确证标准的建立
PTH(1-34)第8、18位为Met,易被氧化,而氧化后的PTH(1-34)生物活性大大降低。因而需尽早对初步筛选菌株表达的融合蛋白及初步纯化得的目的蛋白进行鉴定,淘汰不合标准重组工程菌。
简便易行的标准是用HPLC主峰的保留时间进行判断,因为PTH(1-34)在不同程度氧化下会出现保留时间各不相同的新峰。如果筛选的重组工程菌表达并最终纯化得到的PTH(1-34)其主峰保留时间不在100±0.03%内则视为该重组工程菌不合格。
因此,本实施例用WHO的PTH(1-34)标准确定主峰图谱,并用过氧化氢对其进行氧化确定氧化峰。
1)HPLC方法及样品浓度、纯度的确定
高效液相色谱仪:Shimadzu LC-2040C 3D(日本Shimadzu公司),色谱柱:InsertsilTM WP300 C18(Shimadzu),乙腈购自Honeywell公司,三氟乙酸(TFA)购自Aladdin公司。
流动相A:含0.1%TFA的100%水溶液;流动相B:含0.1%TFA的乙腈-水(90:10)溶液。检测条件:柱温30℃,214nm紫外检测,上样量20μl;梯度洗脱:0-17min,B:10%-30%;17-30min,B:30%-100%;30-38min,B:100%;38-40min,B:100%-10%;40-50min,B:10%;流速:1ml/min。含量计算方法:精密取对照品适量配制成贮备溶液,分别配制成一系列梯度浓度的标准溶液。取各梯度浓度的标准溶液20ul注入色谱仪,记录色谱图,测量标准溶液中待测组分的峰面积。
样品浓度计算方法:以标准溶液的浓度为横坐标,回归计算标准曲线,其公式为:AR=a×CR+b。式中AR为标准溶液中待测组分的峰面积;CR为标准溶液的浓度;a为标准曲线的斜率;b为标准曲线的截距。再取供试品溶液,注入色谱仪,记录色谱图,测量供试品溶液中待测组分的峰面积,按下式计算其浓度:CS=(AS-b)/a。式中AS为供试品溶液中待测组分的峰面积;CS为供试品溶液的浓度;b符号的意义同上。纯度计算方法:采用面积归一化法计算各杂质峰面积及其总和,并求出占总峰面积的百分率,溶剂峰不计算在内。采用标准曲线法测定样品含量,面积归一化法计算待测样品浓度。
纯度计算方法:采用面积归一化法计算各杂质峰面积及其总和,并求出占总峰面积的百分率,溶剂峰不计算在内。采用标准曲线法测定样品含量,面积归一化法计算待测样品纯度。
WHO的PTH(1-34)标准品图谱见图4,本发明精纯PTH(1-34)图谱见图5。
2)过氧化氢氧化PTH(1-34)
取样品溶液50μl分别加入100μl未稀释的3%过氧化氢溶液,稀释了10倍、30倍、60倍浓度的过氧化氢溶液,混匀,放置1-2h,在10000rmp离心5min取上清注入液相色谱仪,按上述HPLC方法进行,PTH(1-34)氧化色谱图如图6。由图6所示,PTH(1-34)依然保留,但大幅减少,同时分别在20.254,20.581及20.733分时出现了三个氧化峰。
实施例3融合蛋白的初步纯化、目的蛋白PTH(1-34)纯化及性质初步鉴定
根据纯化标签性质用Ni-NTA对表达的融合蛋白进行纯化。可溶表达的用破菌上清结合填料,包涵体则加入终浓度为8mol/L尿素溶解后结合填料,根据填料生产厂家推荐的洗涤步骤进行杂蛋白洗涤,然后用洗脱液洗脱融合蛋白。洗脱液稀释后(包涵体洗脱液复性后)再结合相应填料,分别用对应的蛋白酶进行填料上酶切,收集流穿液,用10%SDS-PAGE及HPLC(对照实施例2)初步鉴定目的蛋白性质。
通过本实施例的实验,发现包涵体表达的融合蛋白在纯化后PTH(1-34)与WHO标准品HPLC色谱一致,见图6。可溶形式表达的融合蛋白在纯化后PTH(1-34)出现三个峰,未氧化峰仅占3.47%,见图7;最大峰与对照样品的氧化峰一致,见如图8所示,可溶表达的PTH(1-34)经最终纯化后得到的主要为PTH(1-34)的氧化峰。因此,PTH(1-34)不宜通过可溶形式表达。本实施例展示的是用精纯样品制作的HPLC图。
实施例4:重组PTH(1-34)工程菌的优化构建
通过对实施例1构建的重组载体表达的融合蛋白进行总结得初步结果如表2,
表2各重组工程菌表达情况
可溶表达的融合蛋白,其性质如实施例3的发现,即得到的目的蛋白PTH(1-34)为氧化型;如为包涵体表达,需对融合蛋白完全复性才能进行蛋白酶酶切,也得走现有目的蛋白制备的老路,在纯化过程还得加上反相纯化步骤,在制备过程中使用大量有机溶剂。
为了克服现有技术的弊端,本申请对重组PTH(1-34)工程菌的优化构建策略为:重组融合蛋白以较疏松包涵体形式表达,而融合蛋白能在较高浓度尿素存在的情况下复性且被更容易被制备更经济的TEV酶切割。
通过用PCR方法对引导肽前面各元件进行优化,进行多种形式的组合,反复筛选。利用SUMO帮助融合蛋白折叠、增加可溶性的特点;又利用pET28a载体表达量高、表达增强肽进一步提升表达量的特性。通过快速表达抵消SUMO带来的可溶而形成不紧密的包涵体。最终筛选到重组PTH(1-34)工程菌株,即pET28a-表达增强肽-连接肽1-His6-连接肽2-引导蛋白-连接肽3-蛋白酶切位点-PTH(1-34)/BL21(DE3),简称为pET28a-HSTP/BL21(DE3)。整个融合蛋白的氨基酸序列为SEQ ID NO:12。
实施例5:重组PTH(1-34)工程菌pET28a-HSTP/BL21(DE3)的60L发酵
1)一级摇瓶:
从-80℃冰箱拿出冻存的实施例4构建的pET28a-HSTP/BL21(DE3)工程菌甘油管,解冻后,在无菌操作台上,移取10μl摇匀的甘油种子液(接种量1‰),接种于10ml含50μg/ml卡那霉素(50ppm)LB培养基的带盖三角瓶中(100ml三角瓶),恒温震荡摇床37℃,220rpm,培养5-6h(OD600nm值0.3-0.8)。
2)二级摇瓶
在无菌操作台上,打开已灭菌的含200ml LB培养基的带滤器接种瓶,移取4ml一级种子液(0.5‰)于该接种瓶中,在酒精灯火焰旁接好接种管,将接种瓶中含一级种子菌的LB培养基,压入8L含50μg/ml卡那霉素(50ppm)的已灭菌LB培养基发酵罐中(15L发酵罐),设置37℃,100rpm,空气流量8L/min,培养16-17h(OD600nm值2-5)。
3)上罐发酵
将8L二级种子液压入含50μg/ml卡那霉素的TB培养基中,种后体积60L(种量13.3%),开始发酵培养。诱导前:37℃、pH7.20、溶氧DO=40%培养5h,过程中流加20%的甘油溶液;
诱导后:加入0.50mmol/L终浓度的IPTG,37℃、pH7.20、DO=40%诱导培养3-4h,过程中流加20%的甘油溶液,取诱导0h、1h、2h、3h、4h发酵液做SDS-PAGE电泳,图9,凝胶灰度扫描确定重组蛋白表达率。培养结束后,发酵液于6500rpm、14℃离心12min,收集沉淀于-20℃冰箱保存。
三批发酵结果统计如下:
实施例6 PTH(1-34)的初步纯化
选择Ni-NTA初步纯化,破菌菌体湿重每100g填料用量为10-20ml。采用纯水清洗填料2-5个体积后,采用含有8mol/L尿素的A液平衡填料,然后将上述溶解的包涵体与填料混合,轻轻混匀10-20min,去掉未结合的蛋白,然后还用含有20-25mmol/L咪唑、8mol/L尿素的A液进一步去除填料上的杂蛋白。然后加入适量的带有His标签的TEV酶(1g包涵体加入约0.1ml的酶)进行柱上酶切,酶切液为含有2mol/L尿素的A液,酶切液用量为填料的1-2倍,酶切条件为4-25℃酶切2-5h,酶切过程中垂直混悬确保酶与融合蛋白充分接触,使得酶切充分,酶切的同时完成柱上复性。酶切结束后收集上清用于后续纯化。酶切条件摸索如图10所示,融合蛋白能在比较宽松的条件被TEV酶切割。
实施例7 PTH(1-34)的精制纯化
粗纯样品先进行阳离子交换层析,疏水层析,然后进行阴离子以去除内毒素,收集流穿。
通过此步纯化可获得纯度超过99.5%(图5)的PTH(1-34)1500mg以上。
实施例8 PTH(1-34)的生物学活性测试
取UMR-106细胞消化,800rpm离心3min,重悬计数,将此细胞悬液稀释至1×105个细胞/ml。以100μl/孔均匀的接种至96孔板上,过夜培养。用稀释液(不完全DMEM培养基+0.1%BSA+IBMX(BBI LIFE SCIENCES))将样品(含礼来公司特立帕肽对照品(Lilly))稀释至400ng/ml,继续从400ng/ml往下4倍稀释7个点作为给药浓度,样品浓度即为:400ng/ml、100ng/ml、25ng/ml、6.25ng/ml、1.56ng/ml、0.39ng/ml、0.098ng/ml。取出细胞板,用不完全培养基洗一次,100μl/孔。将刺激液加入96孔板细胞中,100μl/孔,37℃,5%CO2孵育45min,每个浓度梯度做3复孔。刺激完毕后,用DMEM不完全培养基洗1次,100μl/孔。加入裂解液130μl/孔,-80度冻融一次,将同浓度上清吹打后取出混合,4000rpm,离心5min,用于cAMP检测。采用R&D Systems公司的cAMP试剂盒(Cat No.SKGE002B)检测,操作步骤按照说明书试剂盒进行,注意每一次孵育温度控制在20℃。
生物学活性(U/ml)=标准品标示的效价*待测样品预稀释倍数/标准品预稀释倍数*待测样品EC50/标准品EC50。
比活性(U/mg)=标准品标示的效价*样品生物学活性/样品浓度。
结果如图11所示。由图及表3看出,PTH(1-34)(20190802-3-0及20190802-3-1批)的生物学活性不低于对照品礼来特立帕肽。
表3 Lilly公司特立帕肽与本申请PTH(1-34)样品细胞活性比较
比活性(U/mg) | PTH(1-34)样品/lilly样品 | |
Lilly | 10000 | \ |
20190802-3-0 | 10049 | 1.0049 |
20190802-3-1 | 12285 | 1.2285 |
实施例9 PTH(1-34)的圆二色谱测定
委托上海中科新生命生物科技有限公司进行并与WHO标准品进行对照。结果见图12。图形及数据显示,本申请的PTH(1-34)(20190627批)与WHO标准品的二级结构一致。
实施例10 PTH(1-34)的肽图分析
高效液相色谱仪:Shimadzu LC-2040 C 3D(Shimadzu)。色谱柱:Agilent ZORBAX300SB-C18(Agilent),乙腈(Honeywell)公司,三氟乙酸(TFA)(Aladdin)。流动相A:含0.1%TFA的100%水溶液;流动相B:含0.1%TFA的乙腈溶液。缓冲液:1%NH4HCO3、50%冰醋酸溶液。
样品前处理:取供试品溶液及对照品溶液(均为没1ml中含1mg蛋白的溶液),分别用1%的碳酸氢铵溶液置换缓冲液4-5次,浓缩至浓度为1mg/ml的蛋白溶液。取置换缓冲液的供试品溶液和对照品溶液,按1:50(mg/mg)加入胰蛋白酶溶液(Promega)(胰蛋白酶20μg加入20μl buffer溶解),在37℃保温24h,按1:10加入50%冰醋酸溶液,以每分钟10000rmp离心5min,取上清溶液注入液相色谱仪,梯度洗脱,记录色谱图。将供试品溶液与对照品溶液的图谱进行比较,即得。
色谱条件:柱温40℃,214nm紫外检测,对照品和待测样品分别上样20μl;梯度洗脱:0-5min,B:0%-10%;5-40min,B:10%-30%;40-45min,B:30%-40%;45-47min,B:40%-60%;47-50min,B:60%-0%;50-55min,B:0%;流速:1ml/min。
结果如图13所示,由图看出,PTH(1-34)(20190410批)与WHO标准品完全一致。
虽然,上文中已经用一般性说明及具体实施例对本申请作了详尽的描述,但在本申请基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本申请精神的基础上所做的这些修改或改进,均属于本申请要求保护的范围。
序列表
<110> 重庆艾力彼生物科技有限公司
<120> 用于表达甲状旁腺素PTH的融合蛋白及重组质粒、重组工程菌
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Asn Asn Asn Asp Leu Phe Gln
1 5
<210> 2
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Asp Arg Arg Arg Phe Ile Lys Gly Ser
1 5 10
<210> 3
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ser Leu Ser Arg Arg Gln Phe Ile Gln
1 5 10
<210> 4
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Lys Tyr Leu Leu Pro Thr
1 5
<210> 5
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Asn His Lys Val His Met
1 5
<210> 6
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 7
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210> 8
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Asn Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 9
<211> 34
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe
<210> 10
<211> 102
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
agcgttagcg aaatccaact gatgcacaac ctgggtaaac acctgaactc tatggaacgt 60
gttgaatggc tgcgtaaaaa actgcaggac gttcacaact tc 102
<210> 11
<211> 207
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atgcctagtg ttcaagaggt tgaaaaactc cttcatgttc tcgatcgcaa cggtgacggc 60
aaggtttctg ccgaggagtt gaaagccttc gctgatgatt caaaatgtcc tctggactcc 120
aataagatca aggctttcat taaggaacac gataaaaaca aggatggcaa gcttgatttg 180
aaagaactcg tttcgatttt gtcatca 207
<210> 12
<211> 181
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Asn His Lys Val His Met Asn Trp Ser His Pro Gln Phe Glu Lys
1 5 10 15
Ser Ser Gly Ser Ser His His His His His His Gly Gly Ser Gly Gly
20 25 30
Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro Glu
35 40 45
Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser Ser
50 55 60
Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu Met
65 70 75 80
Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg Phe
85 90 95
Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Ala Pro Glu Asp Leu
100 105 110
Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile Gly
115 120 125
Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Asn Leu
130 135 140
Tyr Phe Gln Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys
145 150 155 160
His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln
165 170 175
Asp Val His Asn Phe
180
Claims (5)
1.一种用于表达重组甲状旁腺素PTH的融合蛋白,其特征在于,所述融合蛋白依次由表达增强肽、连接肽1、His6、连接肽2、引导蛋白、连接肽3、蛋白酶切位点和甲状旁腺素PTH1-34组成;
所述融合蛋白的氨基酸序列为SEQ ID NO:12。
2.一种用于表达重组甲状旁腺素PTH的重组质粒,其特征在于,包含骨架质粒和编码如权利要求1所述的融合蛋白的核苷酸序列。
3.如权利要求2所述的重组质粒,其特征在于,所述骨架质粒选自pET系列载体、pGEX系列载体、pMAL系列及pCOLD-SUMO载体中的一种。
4. 如权利要求2或3所述的重组质粒,其特征在于,所述编码融合蛋白的核苷酸序列中编码甲状旁腺素PTH1-34的核苷酸序列为SEQ ID NO:10。
5.一种用于表达甲状旁腺素PTH的重组工程菌,其特征在于,所述重组工程菌含有如权利要求2-4中任一项所述的重组质粒,所述宿主菌选自BL21(DE3)。
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