CN114426962A - t-PA纯化方法 - Google Patents
t-PA纯化方法 Download PDFInfo
- Publication number
- CN114426962A CN114426962A CN202111574380.6A CN202111574380A CN114426962A CN 114426962 A CN114426962 A CN 114426962A CN 202111574380 A CN202111574380 A CN 202111574380A CN 114426962 A CN114426962 A CN 114426962A
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- Prior art keywords
- lysine
- inhibitor
- affinity chromatography
- purification
- expression vector
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- 238000012044 lysine affinity chromatography Methods 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 32
- 239000000523 sample Substances 0.000 claims abstract description 31
- 238000011068 loading method Methods 0.000 claims abstract description 27
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
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Abstract
本发明涉及一种t‑PA纯化方法。该t‑PA纯化方法包括步骤:获得含有t‑PA与t‑PA抑制剂形成的非共价复合物的上样液;及将上样液进行赖氨酸亲和层析,制得纯化的t‑PA。该t‑PA纯化方法,通过将t‑PA与t‑PA抑制剂形成的非共价复合物,和赖氨酸亲和层析的结合使用,能够实现t‑PA的一步层析纯化,并且得到纯度高的t‑PA。一步纯化有利于提高纯化的回收率,以及便于工业化生产,降低生产成本。
Description
技术领域
本发明涉及蛋白纯化领域,特别是涉及一种t-PA纯化方法。
背景技术
机体中的纤溶酶原激活剂有两种,分别是组织型纤溶酶原激活剂t-PA(Tissueplasminogen activator)与尿激酶型纤溶酶原激活剂u-PA(Urokinase plasminogenactivator)。它们均具有激活纤溶酶原成为纤溶酶的特性,因而可被应用于因血栓引起的多种疾病的治疗。其中u-PA由于具有较强的非特异性,既能激活血液凝块中的纤溶酶原,也可激活血液循环中流动的纤溶酶原,有较高的溶血风险。另经研究表明,动物细胞高表达的u-PA其治疗效果不如t-PA。相比之下,t-PA具有较高特异性,只选择性激活血液凝块中的纤溶酶原,不会产生溶血风险,治疗效果也更为理想,故t-PA成为了临床上重要的溶栓治疗药物。
由于t-PA为静脉注射给药,其临床用量比一般生物蛋白要大得多,用于急性心肌梗死的常规剂量为1.25毫克/千克体重,按成年人平均体重50千克一天给药一次计算,给药剂量为62.5毫克/人/天。然而正常细胞能产生的t-PA的量远不能满足临床使用需要,人黑色素瘤细胞的t-PA表达量也只有1.4毫克/升,高密度灌流培养的上皮细胞的t-PA表达量为5毫克/升~10毫克/升,传统的重组CHO细胞(Chinese hamster ovary cells)的t-PA表达量能达120毫克/升。要纯化得到成人一剂量的t-PA,在理想的回收率无损失的情况下,需要人黑色素瘤细胞发酵液44.6升,或上皮细胞发酵液6.25升,或传统的重组CHO细胞发酵液521毫升,可见需要的发酵液量很大。但实际上不可能达到回收率无损失的情况,所需要的发酵液更多,提高t-PA的生产和纯化效率则很有必要。
而目前t-PA的纯化工艺一般需要3~4步才能实现90%以上的纯度,常见的t-PA纯化策略有:经过蓝色琼脂糖凝胶层析、赖氨酸亲和层析和凝胶过滤层析三步层析,或经过阳离子交换层析、赖氨酸亲和层析和疏水层析三步层析,或经过锌离子鳌合层析、伴刀豆球蛋白A亲和层析和凝胶过滤层析三步层析等。这些t-PA的纯化工艺复杂,步骤繁琐,不利于提高纯化回收率及工业化生产。
发明内容
基于此,有必要提供一种t-PA纯化方法,以简便快速地获得大量高纯度的t-PA,降低工业化生产成本。
一种t-PA纯化方法,包括以下步骤:
获得含有t-PA与t-PA抑制剂形成的非共价复合物的上样液;及将上样液进行赖氨酸亲和层析,制得纯化的t-PA。
上述的t-PA纯化方法,通过将t-PA与t-PA抑制剂形成的非共价复合物,和赖氨酸亲和层析的结合使用,能够实现t-PA的一步层析纯化,并且得到纯度高的t-PA。一步纯化有利于提高纯化的回收率,以及便于工业化生产,降低生产成本。
在其中一个实施例中,t-PA抑制剂选自α-抗胰蛋白酶、牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-1、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3、神经源性丝氨酸蛋白酶抑制剂和胶质细胞源性连接蛋白中的至少一种。
在其中一个实施例中,制备上述非共价复合物的步骤包括:构建共表达t-PA和t-PA抑制剂的表达载体;及将该表达载体转染至宿主细胞中表达,制备上述非共价复合物。
在其中一个实施例中,构建上述表达载体的步骤包括:将插入编码t-PA的核苷酸序列的表达载体和插入编码t-PA抑制剂的核苷酸序列的表达载体连接成双表达框表达载体。
在其中一个实施例中,上述宿主细胞为哺乳动物细胞。
在其中一个实施例中,上述宿主细胞为中国仓鼠卵巢细胞。
在其中一个实施例中,上述上样液中t-PA的浓度大于200μg/mL。
在其中一个实施例中,将上样液进行赖氨酸亲和层析的步骤包括:
上样:采用平衡液平衡赖氨酸亲和层析柱后上样;
淋洗:上样结束后,采用淋洗液淋洗赖氨酸亲和层析柱;
洗脱:在淋洗结束后,采用洗脱液洗脱赖氨酸亲和层析柱,收集洗脱流出液。
在其中一个实施例中,洗脱步骤结束后,采用含有0.05mol/L~0.2mol/LNaOH的溶液清洗赖氨酸亲和层析柱。
在其中一个实施例中,所述平衡液包括pH为5.0~8.0的缓冲溶液;所述淋洗液包括含有0.5mol/L~2.0mol/L的钠盐或0.5mol/L~2.0mol/L的钾盐的pH为5.0~8.0的缓冲溶液;所述洗脱液包括含有0.1mol/L~2mol/L的具有赖氨酸结合位点的物质的pH为3.5~7.5的缓冲溶液,所述具有赖氨酸结合位点的物质选自精氨酸、赖氨酸和ε-氨基己酸中至少一种。
在其中一个实施例中,上述缓冲溶液为磷酸缓冲液,也可以是其他能起缓冲作用的溶液。
在其中一个实施例中,上述洗脱液包括:0.1mol/L~2mol/L的具有赖氨酸结合位点的物质、40mmol/L~60mmol/L的NaH2PO4和0%(w/v)~0.02%(w/v)的Tween-80,上述具有赖氨酸结合位点物质选自精氨酸、赖氨酸和ε-氨基己酸中的一种;上述洗脱液的pH为3.5~7.5。
在其中一个实施例中,上述洗脱液包括:0.5mol/L的精氨酸、50mmol/L的NaH2PO4和0.01%(w/v)的Tween-80,洗脱液的pH为7.5。
在其中一个实施例中,上述淋洗液包括:0.5mol/L~2mol/L的NaCl、40mmol/L~60mmol/L的NaH2PO4和0%(w/v)~0.02%(w/v)的Tween-80,该淋洗液的pH为3.5~7.5。在其中一个实施例中,上述淋洗液包括:1mol/L的NaCl、50mmol/L的NaH2PO4和0.01%(w/v)的Tween-80,淋洗液的pH为7.5。
在其中一个实施例中,上述平衡液包括:40mmol/L~60mmol/L的NaH2PO4和0%(w/v)~0.02%(w/v)的Tween-80,该平衡液的pH为5.0~8.0。
在其中一个实施例中,上述平衡液包括:50mmol/L的NaH2PO4和0.01%(w/v)的Tween-80,平衡液的pH为7.5。
附图说明
图1为实施例1中赖氨酸亲和层析所得产物的电泳结果图;
图2为对比例1中苯甲脒亲和层析所得产物的电泳结果图;
图3为对比例2中蓝色琼脂糖凝胶层析所得产物的电泳结果图;
图4为对比例3中阳离子层析所得产物的电泳结果图。
以上各图中,上样流出液为上样步骤收集的流出液,淋洗流出液为淋洗步骤收集的流出液,洗脱流出液为洗脱步骤收集的流出液,清洗流出液为在位清洗步骤收集的流出液。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似改进,因此本发明不受下面公开的具体实施例的限制。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本文所述的“w/v”是指质量体积比。所述的“v/v”是指体积比。
本申请一实施方式提供了一种t-PA纯化方法,包括步骤S1和步骤S2:
步骤S1:获得含有t-PA与t-PA抑制剂形成的非共价复合物的上样液。
在其中一些实施例中,t-PA抑制剂选自α-抗胰蛋白酶、牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-1、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3、神经源性丝氨酸蛋白酶抑制剂和胶质细胞源性连接蛋白中的至少一种。具体地,t-PA是丝氨酸蛋白酶,可以理解的是,t-PA抑制剂则为丝氨酸蛋白酶抑制剂(Serine protease inhibitor,SPI)。t-PA与上述t-PA抑制剂形成的非共价复合物结构松散。若采用抑制作用过强的t-PA抑制剂会与t-PA形成共价复合物,增加t-PA下游纯化的难度。
在其中一些实施例中,t-PA具有如SEQ ID NO:1所示的氨基酸序列。
在其中一些实施例中,t-PA抑制剂为牛胰蛋白酶抑制剂,具有如SEQ IDNO:2所示的氨基酸序列。
在其中一些实施例中,制备上述非共价复合物的方法包括步骤S11和步骤S12:
步骤S11:构建共表达t-PA和t-PA抑制剂的表达载体。
在其中一些实施例中,构建上述表达载体的步骤包括:将插入编码t-PA的核苷酸序列的表达载体和插入编码t-PA抑制剂的核苷酸序列的表达载体连接成双表达框表达载体。在其中一个实施例中,上述双表达框表达载体采用包括如下步骤的方法制备得到:将编码t-PA的核苷酸序列插入pXC17.4表达载体中,得到pXC17.4-tPA;将编码所述t-PA抑制剂的核苷酸序列插入pXC18.4表达载体,得到pXC18.4-SPI;将pXC17.4-tPA和pXC18.4-SPI用NotI和PvuI进行酶切,连接成同时表达tPA和SPI的双表达框表达载体pXC-tPA-SPI。
步骤S12:将该表达载体转染至宿主细胞中表达,制备上述非共价复合物。
在其中一些实施例中,上述宿主细胞为哺乳动物细胞。进一步地,上述宿主细胞选自大鼠、小鼠、仓鼠、豚鼠、猴或人的细胞中的一种。更进一步地,上述宿主细胞为中国仓鼠卵巢细胞(CHO细胞,Chinese hamster ovary cells)。可选地,上述宿主细胞为CHO-K1细胞。CHO-K1细胞是未经改造的野生型CHO细胞,该细胞株培养条件简单,比较容易转染,适合实验或生产使用。可以理解的是,在其他一些实施例中,所使用的细胞不限于是CHO-K1细胞,其他适合实验或生产用的哺乳动物细胞均可使用。相对于酵母细胞需要加入醇氧化酶启动子进行诱导表达,以及细菌(如大肠杆菌)需要加入乳糖进行诱导表达,本发明采用哺乳动物细胞作为宿主细胞,可以无需诱导而实现目标蛋白的组成型表达,从而避免了额外添加诱导剂导致的细胞生长或代谢抑制,进而影响蛋白的翻译后修饰。本发明提供的该实施方式安全性高,更利于目标蛋白后续的纯化继而作为药物使用。
在其中一些实施例中,上述上样液中t-PA的浓度大于200μg/mL。具体地,将步骤S11中的双表达框表达载体转染至宿主细胞后,由于t-PA与t-PA抑制剂均会表达,t-PA抑制剂的存在避免了过量表达的t-PA引起的细胞毒性,不会对细胞的存活造成明显压力,极大地提高了细胞的t-PA产量,提高了上样液中t-PA的浓度,提高了生产效率。
步骤S2:将上样液进行赖氨酸亲和层析,制得纯化的t-PA。
具体地,亲和层析属于一种吸附色谱,其吸附作用主要是靠生物分子对它的互补结合体(配基)的生物识别能力进行选择性分离的一种色谱分离技术。选择赖氨酸作为配体对t-PA进行吸附,即能保证对t-PA的吸附能力强,又能通过淋洗去除与t-PA非特异性结合的杂质,且需要收集t-PA时能将t-PA洗脱下来。
在其中一些实施例中,将上样液进行赖氨酸亲和层析,包括步骤S21、步骤S22和步骤S23:
步骤S21:上样:采用平衡液平衡赖氨酸亲和层析柱后上样。
在其中一些实施例中,上述平衡液包括pH为5.0~8.0的缓冲溶液。在一个可选的具体实施例中,上述平衡液可以是但不限于磷酸缓冲液。
在其中一些实施例中,上述平衡液包括:40mmol/L~60mmol/L的NaH2PO4和0%(w/v)~0.02%(w/v)的Tween-80,该平衡液的pH为5.0~8.0。采用上述平衡液平衡赖氨酸亲和层析柱的目的是减少杂质在层析柱上的非特异性吸附。加入Tween-80的目的是抑制t-PA在容器壁上的吸附,减少t-PA的损失,提高t-PA的回收率。可以理解的是,在一些实施例中Tween-80不是必须添加的。
进一步地,上述平衡液包括:50mmol/L的NaH2PO4和0.01%(w/v)的Tween-80,该平衡液的pH为7.5。
在一个可选的具体示例中,上述平衡液由50mmol/L的NaH2PO4、0.01%(w/v)的Tween-80和水组成,该平衡液的pH为7.5。
步骤S22:淋洗:上样结束后,采用淋洗液淋洗赖氨酸亲和层析柱。
在其中一些实施例中,所述淋洗液包括含有0.5mol/L~2.0mol/L的钠盐或0.5mol/L~2.0mol/L的钾盐的pH为5.0~8.0的缓冲溶液。
在其中一些实施例中,上述淋洗液包括:0.5mol/L~2mol/L的NaCl、40mmol/L~60mmol/L的NaH2PO4和0%(w/v)~0.02%(w/v)的Tween-80,该淋洗液的pH为5.0~8.0。采用上述淋洗液淋洗赖氨酸亲和层析柱的目的是除去上样液中的杂蛋白,杂蛋白包括与t-PA共表达的t-PA抑制剂以及上样液中混有的其他一些生产过程中产生的非t-PA的蛋白。在淋洗过程中可以有效地洗去没有与赖氨酸结合的蛋白。
进一步地,上述淋洗液包括:1mol/L的NaCl、50mmol/L的NaH2PO4和0.01%(w/v)的Tween-80,该淋洗液的pH为7.5。
在一个可选的具体示例中,上述淋洗液由1mol/L的NaCl、50mmol/L的NaH2PO4、0.01%(w/v)的Tween-80和水组成,该淋洗液的pH为7.5。
步骤S23:洗脱:在淋洗结束后,采用洗脱液将赖氨酸亲和层析柱上结合的目的蛋白t-PA进行洗脱,收集洗脱流出液。
在其中一些实施例中,所述洗脱液包括含有0.1mol/L~2mol/L的具有赖氨酸结合位点的物质的pH为3.5~7.5的缓冲溶液,所述具有赖氨酸结合位点的物质选自精氨酸、赖氨酸和ε-氨基己酸中至少一种。
在其中一些实施例中,上述洗脱液包括:0.1mol/L~2mol/L的具有赖氨酸结合位点的物质、40mmol/L~60mmol/L的NaH2PO4和0%(w/v)~0.02%(w/v)的Tween-80,上述具有赖氨酸结合位点的物质选自精氨酸、赖氨酸和ε-氨基己酸中的一种;上述洗脱液的pH为3.5~7.5。采用上述洗脱液清洗赖氨酸亲和层析柱,能够有效地将赖氨酸层析柱上吸附的t-PA洗脱下来,t-PA的回收率高。此外,如上所述,吐温的作用是避免t-PA在容器上吸附,吐温的加入可以提升收率。
选用精氨酸、赖氨酸和ε-氨基己酸中的一种具有赖氨酸结合位点的物质加入洗脱液,目的是加入与t-PA能产生竞争作用的分子,该竞争性分子与层析柱上的赖氨酸结合,使t-PA从赖氨酸亲和层析柱上分离,达到洗脱的目的。
进一步地,上述洗脱液包括:0.5mol/L的精氨酸、50mmol/L的NaH2PO4和0.01%(w/v)的Tween-80,上述洗脱液的pH为7.5。
在一个可选的具体示例中,上述洗脱液由0.5mol/L的精氨酸、50mmol/L的NaH2PO4、0.01%(w/v)的Tween-80和水组成,上述洗脱液的pH为7.5。
在其中一些实施例中,在采用洗脱液清洗赖氨酸亲和层析柱之后,还包括清洗赖氨酸亲和层析柱使其再生再利用的步骤。在其中一些实施例中,采用含有0.05mol/L~0.2mol/L的NaOH溶液对赖氨酸亲和层析柱进行清洗。在一个可选的具体示例中,采用0.1mol/L的NaOH对赖氨酸亲和层析柱进行清洗。
上述的t-PA纯化方法,通过将t-PA与t-PA抑制剂形成的非共价复合物,和赖氨酸亲和层析的结合使用,能够实现t-PA的一步层析纯化,并且得到纯度高的t-PA。一步纯化有利于提高纯化的回收率,以及便于工业化生产,降低生产成本。同时结合本发明的t-PA的生产方法和赖氨酸亲和层析中的具体步骤,能够利用较少的人力物力就得到浓度较高且纯度较高的t-PA,为临床使用提供了极大的资源和方便。
本发明所述技术,通过Lysine HyperD纯化tPA,产率可达361.5mg/L,远远高于现有技术的产率。根据现有报道的最高表达量,就算按照整个纯化流程的回收率为100%计算,其产率也不超过120mg/L,由于纯化的收率不可能达到100%,因此,现有技术的产率不可能超过120mg/L。本发明所述纯化后的产率是现有技术的3倍以上,远远高于现有技术的产率。
具体实施例
以下结合具体实施例进行详细说明。以下实施例如未特殊说明,则不包括除不可避免的杂质外的其他组分。实施例中采用试剂和仪器如非特别说明,均为本领域常规选择。实施例中未注明具体条件的实验方法,按照常规条件,例如文献、书本中所述的条件或者生产厂家推荐的方法实现。
实施例1
1.获得上样液
1)质粒构建
全基因合成t-PA的DNA序列,所用密码子以CHO细胞的偏好密码子进行优化,5’和3’的限制性酶切位点分别为HindIII和EcoRI,氨基酸序列如表1中SEQ ID NO:1所示(36~562为成熟肽)。
全基因合成丝氨酸蛋白酶抑制剂(Serine protease inhibitor,SPI),所用密码子以CHO细胞的偏好密码子进行优化,5’和3’的限制性酶切位点分别为HindIII和EcoRI。氨基酸序列如表1中SEQ ID NO:2所示。
表1
其中,表1中的简并碱基如表2所示。
表2
简并碱基 | 正常碱基 |
R | A/G |
Y | C/T |
M | A/C |
K | G/T |
S | G/C |
W | A/T |
H | A/T/C |
B | G/T/C |
V | G/A/C |
D | G/A/T |
N | A/T/C/G |
将t-PA的序列插入至pXC17.4表达载体中,构建单独表达t-PA的表达质粒pXC17.4-t-PA;将SPI的序列插入pXC18.4表达载体,构建单独表达SPI的表达质粒pXC18.4-SPI。将pXC17.4-t-PA和pXC18.4-SPI用NotI和PvuI酶切连接成一个同时表达t-PA和SPI的双表达框质粒pXC-t-PA+SPI。
2)细胞池构建
将pXC-tPA+SPI质粒通过电穿孔转染至经悬浮无血清驯化的CHO-K1细胞中。转染后用含25μmol/L的甲硫氨酸亚砜胺(MSX)的CD CHO培养基对转染后的细胞进行加压筛选,每3天更换一次培养基,直至细胞活率恢复至90%以上,撤去MSX。
3)细胞培养
将筛选后的细胞池以约0.5×106cells/mL接种至含60mL Dynamis培养基的250mL三角摇瓶中,培养条件:37℃,140RPM,5%(v/v)CO2,85%湿度。从第3天起,每天流加补料培养基3%(v/v)Cell Boost 7a和0.3%(v/v)Cell Boost7b,并将葡萄糖控制在5g/L~8g/L的浓度,培养至第11天。在2000rpm的条件下离心10min收获上清,再经0.22μm滤膜过滤后得上样液,保存于2℃~8℃。
2.赖氨酸亲和层析(层析填料:Lysine HyperD)
赖氨酸亲和层析包括如下操作步骤:
1)平衡
用平衡液冲洗赖氨酸亲和层析柱至基线平稳,该平衡液由50mmol/L的NaH2PO4、0.01%(w/v)的Tween-80和水组成,调节该平衡液的pH至7.5。
2)上样
取步骤1中得到的上样液(t-PA浓度大于200μg/mL)上样,上样液中的总蛋白浓度为0.5mg/mL。
3)淋洗
用淋洗液淋洗赖氨酸亲和层析柱至基线平稳,该淋洗液由1mol/L的NaCl、50mmol/L的NaH2PO4、0.01%(w/v)的Tween-80和水组成,调节该淋洗液的pH为7.5。
4)洗脱
用洗脱液洗脱赖氨酸亲和层析柱至基线平稳。该洗脱液由0.5mol/L的精氨酸、50mmol/L的NaH2PO4、0.01%(w/v)的Tween-80和水组成,洗脱液的pH为7.5。收集洗脱流出液。
5)在位清洗(CIP,cleaning in place)
用0.1mol/L的NaOH对赖氨酸亲和层析柱进行清洗,使其可再生再利用。
3.纯化产物检测
上述层析所得洗脱收集物均采用如下SDS-PAGE方法进行目的条带与纯度的检测:
1)样品处理:取各待检样液稀释至合适浓度,加入4×Loading buffer(非还原)制备获得浓度为0.1μg/μL的蛋白样品,置金属加热仪上100℃热处理10min,冷却后12000RPM离心10min。
2)电泳:取20μL处理后样品加入4%~20%SDS-PAGE胶(梯度胶)泳道内,采用140V恒压电泳约1h至指示剂条带距离胶下端0.5cm处。
3)染色与脱色:电泳结束后取出SDS-PAGE胶,置染色脱色仪中进行染色与脱色处理。
4)成像:采用凝胶成像仪成像并拍照保存。
赖氨酸亲和层析的结果如图1所示。从图1中可以看出,收集到的洗脱流出液的泳道中只有目标条带,没有肉眼可见的杂蛋白带存在,说明用此方法纯化得到的t-PA纯度较高。且经测定,目标产物浓度能达到361.5mg/L。通过此方法能够高效率地获得纯度和浓度都较高t-PA产物。
对比例1
对比例1的步骤与实施例1大致相同,其不同在于对比例1的步骤2中采用苯甲脒亲和层析替代赖氨酸亲和层析,且各缓冲液不同,具体为:
苯甲脒亲和层析(层析填料:Benzamidine Sepharose 4FF)包括如下操作步骤:
1)平衡
用平衡液冲洗苯甲脒层析柱至基线平稳,该平衡液为磷酸盐缓冲液(PBS),平衡液pH为7.4。
2)上样
取4mL步骤1中得到的上样液(t-PA浓度大于200μg/mL)上样,上样液中的总蛋白浓度为0.5mg/mL。
3)淋洗
用淋洗液淋洗苯甲脒亲和层析柱至基线平稳,该淋洗液为磷酸盐缓冲液(PBS),平衡液pH为7.4。收集淋洗流出液。
4)洗脱
用洗脱液冲洗层析柱至基线平稳,该洗脱液由0.1mol/L的精氨酸、0.1mmol/L的NH4HCO3和水组成,调节该洗脱液的pH至8.0。按0.5mL每管收集洗脱流出液。
苯甲脒亲和层析的结果如图2所示。从图2中可以看出,收集到的洗脱流出液的泳道中都包含了目标蛋白,但也都有明显的杂蛋白带出现,说明该纯化方法效果差。
对比例2
对比例2的步骤与实施例1大致相同,其不同在于本对比例步骤2中采用蓝色琼脂糖凝胶层析替代赖氨酸亲和层析,且各缓冲液不同,具体为:
蓝色琼脂糖凝胶层析(层析填料:Capto Blue)包括如下操作步骤:
1)平衡
用平衡液冲洗蓝色琼脂糖凝胶层析柱至基线平稳。该平衡液为磷酸缓冲液(10mmol/L PB),pH为7.0。
2)上样
取4mL步骤1中得到的上样液(t-PA浓度大于200μg/mL)上样,上样液中的总蛋白浓度为0.5mg/mL。
3)淋洗
用淋洗液淋洗蓝色琼脂糖凝胶层析柱至基线平稳。该淋洗液为磷酸缓冲液(10mmol/L PB),pH为7.0。收集淋洗流出液。
4)洗脱
用洗脱液冲洗层析柱至基线平稳,该洗脱液由10mmol/L的PB、0~1mol/L的NaCl和水组成,洗脱液pH为7.0。用该洗脱液按10CV(10倍柱体积)进行线性洗脱,收集洗脱流出液。
5)在位清洗
用0.1mol/L的NaOH对蓝色琼脂糖凝胶层析柱进行清洗。
蓝色琼脂糖凝胶层析的结果如图3所示。从图3中可以看出,收集到的在位清洗流出液所在的泳道出现了大量与上样液几乎同样的目标条带,而收集到的洗脱流出液中并未出现蛋白峰,说明对比例2的纯化方法效果非常差。
对比例3
对比例3的步骤与实施例1大致相同,其不同在于本对比例步骤2中采用阳离子层析替代赖氨酸亲和层析,且各缓冲液不同,具体为:
阳离子层析(层析填料:SP Sepharose HP)包括如下操作步骤:
1)平衡
用平衡液冲洗阳离子层析柱至基线平稳。该平衡液为磷酸缓冲液(10mmol/L PB),pH为7.0。
2)上样
取4mL步骤1中得到的上样液(t-PA浓度大于200μg/mL)上样,上样液中的总蛋白浓度为0.5mg/mL。
3)淋洗
用淋洗液淋洗阳离子层析柱至基线平稳。该淋洗液为磷酸缓冲液(10mmol/L PB),pH为7.0。收集淋洗流出液。
4)洗脱
用洗脱液冲洗阳离子层析柱至基线平稳。该洗脱液由10mmol/L的PB、0~1mol/L的NaCl和水组成,洗脱液pH为7.0。用该洗脱液按10CV(10倍柱体积)进行线性洗脱,按2mL每管收集洗脱流出液。
5)在位清洗
用0.1mol/L的NaOH对阳离子层析柱进行清洗,收集流出液。
阳离子层析的结果如图4所示。从图4中可以看出,收集到的洗脱流出液的泳道中出现了目标条带,但存在明显的杂蛋白带,说明对比例3的纯化方法效果差。
本发明中,以上各实施例中层析各步骤均保持4min的保留时间。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。应当理解的是,在本领域技术人员在本发明提供的技术方案的基础上,通过合乎逻辑的分析、推理或有限的试验得到的技术方案,均在本发明所附权利要求的保护范围内。因此,本发明专利的保护范围应以所附权利要求为准,说明书及附图可以用于解释权利要求的内容。
序列表
<110> 佛山汉腾生物科技有限公司
广州汉腾生物科技有限公司
佛山普津生物技术有限公司
<120> t-PA纯化方法
<160> 2
<170> SIPOSequenceListing 1.0
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Ile Tyr Gln Gln His Gln Ser Trp Leu Arg Pro Val Leu Arg Ser Asn
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Arg Val Glu Tyr Cys Trp Cys Asn Ser Gly Arg Ala Gln Cys His Ser
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Cys Gln Gln Ala Leu Tyr Phe Ser Asp Phe Val Cys Gln Cys Pro Glu
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Glu Asp Gln Gly Ile Ser Tyr Arg Gly Thr Trp Ser Thr Ala Glu Ser
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Gly Ala Glu Cys Thr Asn Trp Asn Ser Ser Ala Leu Ala Gln Lys Pro
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Tyr Ser Gly Arg Arg Pro Asp Ala Ile Arg Leu Gly Leu Gly Asn His
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Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro Trp Cys Tyr Val
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Phe Lys Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys
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Ser Glu Gly Asn Ser Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg
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Gly Thr His Ser Leu Thr Glu Ser Gly Ala Ser Cys Leu Pro Trp Asn
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Gln Ala Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Gly
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Asp Ala Lys Pro Trp Cys His Val Leu Lys Asn Arg Arg Leu Thr Trp
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Ser Gln Pro Gln Phe Arg Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala
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Ser His Pro Trp Gln Ala Ala Ile Phe Ala Lys His Arg Arg Ser Pro
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Trp Glu Asn Leu
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Claims (12)
1.一种t-PA纯化方法,其特征在于,包括以下步骤:
获得含有t-PA与t-PA抑制剂形成的非共价复合物的上样液;及
将所述上样液进行赖氨酸亲和层析,制得纯化的t-PA。
2.根据权利要求1所述的t-PA纯化方法,其特征在于,所述t-PA抑制剂选自α-抗胰蛋白酶、牛胰蛋白酶抑制剂、纤溶酶原激活剂抑制剂-1、纤溶酶原激活剂抑制剂-2、纤溶酶原激活剂抑制剂-3、神经源性丝氨酸蛋白酶抑制剂和胶质细胞源性连接蛋白中的至少一种。
3.根据权利要求1或2所述的t-PA纯化方法,其特征在于,制备所述非共价复合物的步骤包括:
构建共表达t-PA和t-PA抑制剂的表达载体;及
将所述表达载体转染至宿主细胞中表达,制备所述非共价复合物。
4.根据权利要求3所述的t-PA纯化方法,其特征在于,构建所述表达载体的步骤包括:将插入编码t-PA的核苷酸序列的表达载体和插入编码t-PA抑制剂的核苷酸序列的表达载体连接成双表达框表达载体。
5.根据权利要求3所述的t-PA纯化方法,其特征在于,所述宿主细胞为哺乳动物细胞。
6.根据权利要求5所述的t-PA纯化方法,其特征在于,所述宿主细胞为中国仓鼠卵巢细胞。
7.根据要求1~2和4~6中任一项所述的t-PA纯化方法,其特征在于,所述上样液中t-PA的浓度大于200μg/mL。
8.根据要求1~7任一项所述的t-PA纯化方法,其特征在于,所述上样液进行赖氨酸亲和层析的步骤包括:
上样:采用平衡液平衡赖氨酸亲和层析柱后上样;
淋洗:上样结束后,采用淋洗液淋洗赖氨酸亲和层析柱;
洗脱:在淋洗结束后,采用洗脱液洗脱赖氨酸亲和层析柱,收集洗脱流出液。
9.根据权利要求8所述的t-PA纯化方法,其特征在于,所述平衡液包括pH为5.0~8.0的缓冲溶液;所述淋洗液包括含有0.5mol/L~2.0mol/L的钠盐或0.5mol/L~2.0mol/L的钾盐的pH为5.0~8.0的缓冲溶液;所述洗脱液包括含有0.1mol/L~2mol/L的具有赖氨酸结合位点的物质的pH为3.5~7.5的缓冲溶液,所述具有赖氨酸结合位点的物质选自精氨酸、赖氨酸和ε-氨基己酸中至少一种。
10.根据权利要求9所述的t-PA纯化方法,其特征在于,所述洗脱液包括:0.1mol/L~2mol/L的具有赖氨酸结合位点的物质、40mmol/L~60mmol/L的NaH2PO4和0%(w/v)~0.02%(w/v)的Tween-80,所述具有赖氨酸结合位点物质选自精氨酸、赖氨酸和ε-氨基己酸中的一种。
11.根据权利要求9或10所述的t-PA纯化方法,其特征在于,所述淋洗液包括:0.5mol/L~2mol/L的NaCl、40mmol/L~60mmol/L的NaH2PO4和0%(w/v)~0.02%(w/v)的Tween-80。
12.根据权利要求9或10所述的t-PA纯化方法,其特征在于,所述平衡液包括:40mmol/L~60mmol/L的NaH2PO4和0%(w/v)~0.02%(w/v)的Tween-80。
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