CN111018965A - 一种重组甲状旁腺素pth(1-34)的纯化方法 - Google Patents
一种重组甲状旁腺素pth(1-34)的纯化方法 Download PDFInfo
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Abstract
本申请公开了一种重组甲状旁腺素PTH的分离纯化方法,所述方法包括:1)细菌破碎与包涵体处理,通过高压匀浆破菌,破菌后高速离心收集沉淀,沉淀经过重悬洗涤后与复溶液混合复溶;2)亲和层析;所述填料选自Ni‑NTA,GST Sepharose和MBP Sepharose中的一种;3)柱上酶切与复性;4)阳离子交换层析和/或疏水层析;5)脱盐。本发明能够高效的从包涵体中提取具有生物学活性的PTH(1‑34)多肽,从而使得PTH(1‑34)的大规模工业化生产成为可能。
Description
技术领域
本发明属于蛋白质分离纯化技术领域,具体涉及一种分离纯化重组甲状旁腺素PTH(1-34)的方法。
背景技术
甲状旁腺素(Parathyroid hormone,PTH)是由84个氨基酸组成的多肽激素,由甲状旁腺上皮细胞合成、储存和分泌。进入血循环的PTH会迅速转变成氨基端和羧基端二个肽段,其中氨基端1-34位氨基酸残基肽段具有完整的PTH生物活性,调节血钙浓度及骨代谢,免疫活性小,而羧基端的肽段为PTH免疫活性多肽,其生物学作用还不是十分清楚。
PTH主要发挥抗骨质疏松的生物学功能,其对骨形成的影响主要是通过作用于骨组织细胞和肾小管上皮细胞而产生的。对于肾小管上皮细胞,PTH主要增加远曲小管的钙的重吸收和通过增加肾脏产生有活性的维生素D间接促进肠道对钙的吸收,共同提高血钙浓度,有利于骨的形成。对于骨组织则既有促进骨吸收,又有促进骨形成作用。
美国Eli Lilly公司早在上世纪九十年代就研发重组人PTH(1-34),并命名为Teriparatide,用于骨质疏松的治疗。国内有一些研发单位和企业正在开发重组人甲状旁腺素,目前上海联合赛尔的产品信立泰已获得生产批件,有些处于临床前研究或临床研究,有些已经进入临床试验阶段。
对于本品的纯化方法,已有专利技术进行公开,如CN102993293A、CN102731643A、CN1212336C等。但这些方法普遍存在需要使用有机溶剂影响多肽活性、多肽易降解、收率低、工艺流程复杂等问题。为解决上述问题,仍有必要对纯化方法进行进一步的研究。
发明内容
针对上述技术问题,本发明公开了一种简便的的纯化方法,能够高效的从包涵体中提取具有生物学活性的PTH(1-34)多肽,从而使得PTH(1-34)的大规模工业化生产成为可能。
本发明公开了一种重组甲状旁腺素PTH的分离纯化方法,所述方法包括:
1)细菌破碎与包涵体处理
通过高压匀浆破菌,破菌后高速离心收集沉淀,沉淀经过重悬洗涤后与复溶液混合复溶
2)根据融合蛋白的标签肽段选择适当亲和层析填料进行亲和层析;所述填料选自Ni-NTA,GST Sepharose和MBP Sepharose中的一种;
3)柱上酶切与复性
其中,酶选自TEV酶、EK酶、Xa因子蛋白酶中的一种;
4)阳离子交换层析和/或疏水层析
阳离子交换层析柱的填料选自SP Sepharose HP、SP Sepharose FF、Capto SP、Capto MMC中的一种;
所述疏水层析柱的填料选自Phynel、Octyl或者Butyl的HP或FF填料中的一种;
5)脱盐。
在根据本发明的一个实施方案中,步骤1)中,步骤1)包括:
菌体与A液以重量:体积比1:5-10的比例混合,并预冷至1-4℃。
在根据本发明的一个实施方案中,步骤1)包括:
1-4℃温度下以60-80Mpa高压匀浆破菌3-5次;破菌后的液体,于4℃,10,000-15,000g条件下离心15-30min,收集沉淀备用。
在根据本发明的一个实施方案中,步骤1)包括:
以重悬液将包涵体重悬,所述包涵体与重悬液的重量:体积比为1:5-10,磁力搅拌器搅拌15-30min,10,000-15,000g离心15-30min,收集沉淀备用;所述重悬液为含有终浓度为0.5%-1.5%的Triton X1000、tween20、tween80,或者sacosyl的A液。
在根据本发明的一个实施方案中,步骤1)包括:
以重量:体积比为1:5-10比例加入复溶液重悬包涵体,混合均匀后,离心并收集上清备用;所述复溶液以A液为溶剂,含有8M尿素、6M盐酸胍或者2mol/L尿素加sacosyl至终浓度((体积百分比浓度))为0.3%的混合液。
在根据本发明的一个实施方案中,步骤2)包括:
以g:ml计,破菌菌体湿重与填料的比例为10:1-2,将复溶的包涵体与填料混匀后以含有20-25mM咪唑、8M尿素的A液进一步去除填料上的杂蛋白;
优选地,所述层析柱填料在与复溶的包涵体混合前先以数个体积的水清洗,再以含有8M尿素的A液平衡。
在根据本发明的一个实施方案中,步骤3)包括:
在亲和加入适量的带有His标签的蛋白酶,进行柱上酶切,酶切液为含有2-4M尿素或盐酸胍的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;其中,以g:ml计,包涵体与酶的重量体积比为10:1。
在根据本发明的一个实施方案中,所述步骤4)包括:
收集亲和层析或疏水层析后的样品以B液稀释3-6倍并调整pH至6.5;然以用C液()线性梯度洗脱,设定洗脱流速5ml/min,洗脱梯度为B液从0到50%,洗脱体积为10个柱体积;其中,所述B液为pH6.5的20mM的磷酸盐缓冲液(PB);所述C液为含有1M NaCl的pH6.5的20mM的磷酸盐缓冲液(PB)。
在根据本发明的一个实施方案中,所述步骤4)包括:
将亲和层析或阳离子交换层析获得的样品与3M的硫酸铵溶液混合至硫酸铵终浓度为1-1.5M;
以D液平衡层析***及层析柱后上样,上样结束后用B液线性梯度洗脱,洗脱流速5ml/min,洗脱梯度为B液从0到100%,洗脱体积为10个柱体积,收集蛋白保存在4℃;所述D液为含有1-1.5M硫酸铵pH6.5的20mM的磷酸盐缓冲液。
在根据本发明的一个实施方案中,所述步骤5)包括:
所述脱盐是通过选自超滤、渗滤、分子筛或脱盐柱中的一种实现的。
本发明的特色和创新之处在于:
采用本发明所述的纯化方法,从表达PTH(1-34)的大肠杆菌工程菌中可以获得纯度大于98%,回收率大于40%的目的蛋白。通过氨基酸序列测定发现其N、C端序列与理论值一致。质谱检测分子量在4117道尔顿左右,与理论值相符。通过细胞实验证实其具有良好的刺激cAMP分泌的能力,不低于Sigma公司购买的标准品,表明其具有良好的生物学活性。
此外,本方法为非HPLC纯化方法,避免了有机溶剂等对蛋白活性的损害。该方法成功实现了柱上酶切,酶切和复性同步进行,大大缩短了纯化流程。该方法操作简单,无需特殊仪器设备和试剂等,有利于工业化扩大生产。
附图说明
图1为PA5055纯化工艺流程图
图2为阳离子层析图;其中流穿样品中有少量杂蛋白,收集洗脱时的高峰用于后续纯化。
图3为疏水层析图;洗脱时前面的峰为杂蛋白,后面的峰为PTH(1-34);
图4为蛋白原液SDS-PAGE电泳结果;
图5为蛋白PTH(1-34)蛋白原液HPLC检测结果。
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
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。
primeSTAR HS DNA聚合酶、DNA分子量标准、限制性内切酶、蛋白分子量标准、DNA连接酶、点突变试剂盒(MutanBest)等为大连宝生物(TakaRa)公司产品;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。
PBS(磷酸二氢钾(KH2PO4)0.2g(国产分析纯),磷酸氢二钠(Na2HPO4·12H2O)2.9g(国产分析纯),氯化钠(NaCl)8.0g(国产分析纯),氯化钾(KCl)0.2g,加水至1000mL,pH7.4);20mol/L PB缓冲液:磷酸二氢钾(KH2PO4)0.2g,磷酸氢二钠(Na2HPO4·12H2O)2.9g,氯化钾(KCl)0.2g,加水至1000mL,pH6.0;氨苄西林、卡那霉素(华北制药);5×蛋白质上样缓冲液:250mmol/L Tris-HCl(pH6.8)、10%(W/V)SDS、0.5%(W/V)溴酚蓝、50%(V/V)甘油、5%(W/V)β-巯基乙醇;谷胱甘肽琼脂糖凝胶4B(美国GE Healthcare公司);Ni-NTA购自Novagen公司。琼脂粉、吐温-20等其余试剂均为国内市场购买。
为了使本发明的目的、技术方案及优点更加清楚明白,以下本室构建的重组工程菌pET28a-PTH(1-34)/BL21(该工程菌株在PTH(1-34)的N端依次引入了His标签、SUMO标签和TEV酶的酶切位点,His标签有利于后续纯化,SUMO标签有利于蛋白折叠和复性,TEV酶切后能保证PTH(1-34)的N端没有残留氨基酸)为例,结合附图及实施例,对本发明进一步详细说明。本发明的纯化工艺流程如图1所示。
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:1的氨基酸序列,将编码DNA密码优化后为SEQ ID NO:2。
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:3)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)。
实施例2: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
若为可溶表达的融合蛋白,则得到的目的蛋白PTH(1-34)为氧化型;如为包涵体表达,则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:4。
实施例3细菌破碎与包涵体的处理
菌体重悬:将上述pET28a-PTH(1-34)/BL21大肠杆菌工程菌,通过高密度发酵,离心收集菌体备用。取菌体50g左右,按重量(g):体积(ml)比1:5-10比例与pH6.0-pH8.5的PBS缓冲液(A液)混匀悬浮,4℃预冷。
高压破菌:使用蒸馏水冲洗高压匀浆机管道,低温循环***开启预冷至1-4℃备用。预冷的悬浮菌液加入高压匀浆机,压力维持在60-80Mpa破菌3-5次,取破菌液涂片结晶紫染色,油镜每个视野下未破碎菌小于1-2个视为破菌完全。
高速离心:破菌后的液体装入离心桶,4℃,10,000-15,000g离心15-30min,收集沉淀备用备用。
包涵体洗涤:采用含有0.5%-1.5%Triton X100的A液按重量:体积比1:5-10比例重悬包涵体,磁力搅拌器搅拌15-30min,10,000-15,000g离心15-30min,收集沉淀备用。
包涵体复溶:采用含有8M尿素的A液按重量:体积比1:5-10比例重悬包涵体,磁力搅拌器搅拌15-30min,10,000-15,000g离心15-30min,收集上清备用。
实施例4 Ni-NTA亲和层析,TEV酶柱上酶切与复性
选择Ni-NTA亲和层析填料进行初步纯化,GST亲和填料不同公司的不同型号产品,本实施例中采用invitrogen公司的Ni-NTA亲和层析填料,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8M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20-25mM咪唑、8M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2M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。
实施例5阳离子交换层析
收集亲和层析的样品,用B液(20mM的PB,pH6.5)稀释3-6倍并调整pH至6.5左右备用。采用B液平衡层析***及5ml的SP HP层析柱,然后进行上样,上样结束后用A液洗脱未结合的杂蛋白。用C液(20mM的PB,1M NaCl,pH6.5)线性梯度洗脱,设定洗脱流速5ml/min,洗脱梯度为B液从0到50%,洗脱体积为10个柱体积,最后用100%的B液洗脱核酸等杂质,收集洗脱下来的蛋白保存在4℃备用。层析图如图2所示,其中流穿样品中有少量杂蛋白,洗脱时有两个峰融合在一起,收集洗脱时的高峰用于后续纯化。
实施例6:疏水层析与置换缓冲液
将上述阳离子交换层析获得的样品,加入3M的硫酸铵溶液至硫酸铵终浓度为1-1.5M,采用D液(20mM的PB,1-1.5M硫酸铵,pH6.5)平衡层析***及5ml Phynel HP层析柱,然后进行上样,上样结束后用A液洗脱未结合的杂蛋白。用B液线性梯度洗脱,设定洗脱流速5ml/min,洗脱梯度为B液从0到100%,洗脱体积为10个柱体积,收集蛋白保存在4℃备用。层析图如图3所示,在洗脱时出现两个峰,前面的峰为杂蛋白,后面的峰为目的蛋白PTH(1-34),两个峰能够有效的得到分离。
脱盐采用50ml的G25脱盐柱直接置换缓冲液,采用的缓冲液为200mM的醋酸钠,150mM NaCl,脱盐结束后取样进行SDS-PAGE鉴定,分装样品并冻存于-80。脱盐后的样品电泳结果如图4所示,样品具有极高的纯度,基本看不到其它杂蛋白。
实施例7:HPLC检测
使用C18柱(购自Agilent公司)对上述纯化的PTH(1-34)蛋白进行纯度检测,用0.1%TFA水溶液平衡柱子,上样5ul样品,0.1%TFA乙腈溶液洗脱,设定柱温60℃,流速0.5ml/min。洗脱程序为:10%~100%B,30min。质谱图如图5所示,结果表明PTH(1-34)纯度在99%左右,达到了生物制品对蛋白类药品的纯度要求。其中,0.1%TFA水溶液的配制:1LI级水加1ml TFA混匀,0.22μm滤膜过滤。0.1%TFA乙腈溶液的配制:1L乙腈加1ml TFA混匀。
实施例8:细胞动物
1)细胞准备:取Saos-2细胞2瓶,弃去培养基,每瓶加入3ml PBS洗涤后弃去。各加胰酶1ml进行消化后加入5ml不完全培养基终止消化。用弯头滴管吹打,使细胞全部脱落后把液体移至15ml离心管中,1500r*5min离心,弃上清,加入1ml完全培养基混匀后进行细胞计数。计数后用完全培养基配制成2×105/ml,200ul/孔加入96孔板,37℃,5%CO2,过夜培养,约28h。
(2)PTH样品及培养基的准备:采用醋酸钠缓冲液(pH4.0)调整PTH样品至250ug/ml。配置完全培养基(McCoy’s培养基+10%胎牛血清+1%HEPES)和测活培养基(0.1%牛血清白蛋白加完全培养基定容至10ml+0.5M IBMX+相应蛋白样品)。
(3)刺激细胞及样品处理:取8个EP管,编号1-7。向2-7管中分别加入测活培养基180ul。1管中加入测活培养基312ul,测定蛋白浓度后计算应加入1管中体积,使得1管中蛋白浓度为4000ng/ml。从1管中取60ul转移至2管中混匀,浓度即为1000ng/ml,从2管中取60ul转移至3管中混匀,以此加到第7管。在细胞板上设有7孔,对应编号1-7,每孔加入150ul蛋白样品刺激,另设一孔为细胞空白,37度刺激15min。实验中设置复孔,并采用Sigma公司购买的PTH标准品作为对照。
刺激结束后,吸弃上清,每孔加入200ul PBS洗涤两遍后每孔加入80ul 0.1M HCL室温放置20Min。完毕后,用枪头吹打几次细胞,从各孔中分别取出液体70ul至EP管中,再每孔加入70ul(0.5M PB溶液+0.1M NaOH溶液)中和PH至7.24左右。1500rpm×10min离心。此为样品。
(4)ELISA操作:采用竞争抑制法检测各刺激样品中cAMP含量,严格按照cayman公司试剂盒说明书操作。
结果表明:采用本方案纯化而得的PTH(1-34)具有刺激Saos-2细胞生成cAMP的生物学活性,且其活性不低于Sigma公司的标准品。
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> 一种重组甲状旁腺素PTH(1-34)的纯化方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 34
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
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> 2
<211> 102
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
agcgttagcg aaatccaact gatgcacaac ctgggtaaac acctgaactc tatggaacgt 60
gttgaatggc tgcgtaaaaa actgcaggac gttcacaact tc 102
<210> 3
<211> 207
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
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> 4
<211> 181
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
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 (10)
1.一种重组甲状旁腺素PTH的分离纯化方法,其特征在于,所述方法包括:
1)细菌破碎与包涵体处理
通过高压匀浆破菌,破菌后高速离心收集沉淀,沉淀经过重悬洗涤后与复溶液混合复溶;
2)亲和层析;所述填料选自Ni-NTA,GST Sepharose和MBP Sepharose中的一种;
3)柱上酶切与复性:
其中,酶选自TEV酶、EK酶、Xa因子蛋白酶中的一种;
4)阳离子交换层析和/或疏水层析:
阳离子交换层析柱的填料选自SP Sepharose HP、SP Sepharose FF、Capto SP、CaptoMMC中的一种;
所述疏水层析柱的填料选自Phynel、Octyl或者Butyl的HP或FF填料中的一种;
5)脱盐。
2.如权利要求1所述的方法,其特征在于,步骤1)中,步骤1)包括:
菌体与A液以重量:体积比1:5-10的比例混合,并预冷至1-4℃。
3.如权利要求1或2所述的方法,其特征在于,步骤1)包括:
1-4℃温度下以60-80Mpa高压匀浆破菌3-5次;破菌后的液体,于4℃,10,000-15,000g条件下离心15-30min,收集沉淀备用。
4.如权利要求1-3中任一项所述的方法,其特征在于,步骤1)包括:
以重悬液将包涵体重悬,所述包涵体与重悬液的重量:体积比为1:5-10,磁力搅拌器搅拌15-30min,10,000-15,000g离心15-30min,收集沉淀备用;所述重悬液为含有终浓度为0.5%-1.5%的Triton X1000、tween20、tween80,或者sacosyl的A液。
5.如权利要求1-4中任一项所述的方法,其特征在于,步骤1)包括:
以重量:体积比为1:5-10比例加入复溶液重悬包涵体,混合均匀后,离心并收集上清备用;所述复溶液以A液为溶剂,含有8M尿素、6M盐酸胍或者2mol/L尿素加sacosyl至终浓度为0.3%的混合液。
6.如权利要求1-5中任一项所述的方法,其特征在于,步骤2)包括:
以g:ml计,破菌菌体湿重与填料的比例为10:1-2,将复溶的包涵体与填料混匀后以含有20-25mM咪唑、8M尿素的A液进一步去除填料上的杂蛋白;
优选地,所述层析柱填料在与复溶的包涵体混合前先以数个体积的水清洗,再以含有8M尿素的A液平衡。
7.如权利要求1-6中任一项所述的方法,其特征在于,步骤3)包括:
在亲和加入适量的带有His标签的蛋白酶,进行柱上酶切,酶切液为含有2-4M尿素或盐酸胍的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;其中,以g:ml计,包涵体与酶的重量体积比为10:1。
8.如权利要求1-6中任一项所述的方法,其特征在于,所述步骤4)包括:
收集亲和层析或疏水层析后的样品以B液稀释3-6倍并调整pH至6.5;然以用C液()线性梯度洗脱,设定洗脱流速5ml/min,洗脱梯度为B液从0到50%,洗脱体积为10个柱体积;其中,所述B液为pH6.5的20mM的磷酸盐缓冲液(PB);所述C液为含有1M NaCl的pH6.5的20mM的磷酸盐缓冲液(PB)。
9.如权利要求1-8中任一项所述的方法,其特征在于,所述步骤4)包括:
将亲和层析或阳离子交换层析获得的样品与3M的硫酸铵溶液混合至硫酸铵终浓度为1-1.5M;
以D液平衡层析***及层析柱后上样,上样结束后用B液线性梯度洗脱,洗脱流速5ml/min,洗脱梯度为B液从0到100%,洗脱体积为10个柱体积,收集蛋白保存在4℃;所述D液为含有1-1.5M硫酸铵pH6.5的20mM的磷酸盐缓冲液。
10.如权利要求1-9中任一项所述的方法,其特征在于,所述步骤5)包括:
所述脱盐是通过选自超滤、渗滤、分子筛或脱盐柱中的一种实现的。
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