Soluble expression and function of von Willebrand factor-A1 in Escherichia coli

doi:10.3969/j.issn.2095-4344.2014.38.015

Abstract

Abstract:

BACKGROUND: von Willebrand factor-A1 (VWF-A1) plays a crucial role in primary hemostasis by mediating blood platelet adhesion to sites of vascular damage under conditions of high shear stress. However, mostly expression of recombinant VWF-A1 in form of inclusion bodied within Escherichia coli (E. coli) that is not conducive to purify and functional study in vitro.
OBJECTIVE: To acquire soluble recombinant VWF-A1 including wild type and its mutation G1324S which can be stable efficiently expressed in E. coli and to verify their biological function.
METHODS: Wild-type VWF-A1 and its mutation G1324S recombinant plasmids were transformed into competent cells DH5α, to screen and extract the plasmids. Then the extracted plasmids were respectively transformed into E. coli BL21 and E. coli M15, to screen monoclonal bacterium. The bacterium was cultured in LB culture medium. The bacteria breaking methods with lysozyme and with ultrasonic were compared. Then, the solubilized proteins were passed over Ni-NTA agarose column to purify the VWF-A1 protein, and the purity and immune activity of purified products were identified with SDS-PAGE and western blot analysis. Finally, the biological function of the purified proteins was assessed by their ability to support flow-dependent platelet adhesion by parallel-plate flow chamber system.
RESULTS AND CONCLUSION: We obtained about 5.77 mg VWF-A1, 6.83 mg VWF-G1324S in culture supernatants per 100 milliliter, respectively, which all showed high purity and immune activity. The data of flow chamber experiment demonstrated that both VWF-A1 and VWF-G1324S could mediate platelet adhesion. As the shear stress increased from 0.1 Pa to 1 Pa, the decline (46.8%) of the number of adhesive cells in G1324S mutation group was higher than that of wild type group (20.5%). This evidence indicated that, the A1 molecule functions were attenuated after mutation. Our research shows that, wild type of VWF-A1 showed a stronger interaction with platelet in shear stress, which is consistent with clinical characteristics.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: von Willebrand Diseases, hemophilia A, blood platelets

CLC Number:

R318

Wang Yi-lu, Liu Xiao-ling, Ding Xiao-ru, Fang Ying. Soluble expression and function of von Willebrand factor-A1 in Escherichia coli[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(38): 6153-6159.

Figures/Tables 2

2.1 两种方法诱导血管性血友病因子A1的可溶性表达使用溶菌酶破碎法：即1.5 mmol/L的IPTG诱导培养 4 h后，经过溶菌酶的处理反复冻融后，得到的上清和沉淀产物(图1A)，从图中可以看出，上清和沉淀中的目的蛋白含量相差很大，诱导后大量的目的蛋白均处于沉淀中，即目的蛋白以其疏水区积聚在一起，几乎都以包涵体方式表达。使用超声波破碎法：即0.2 mmol/L的IPTG诱导表达 6 h后，经过超声波破碎后，得到的上清和沉淀产物(图1B)，此时，上清中的目的蛋白含量与沉淀中的相似，相对于溶菌酶破碎法处理法得到的上清中的目的蛋白的产量有了极大的提升，达到进一步纯化要求。 SDS-PAGE结果显示：两种方法均能实现目的蛋白的成功表达，但超声波破碎法通过降低诱导剂浓度、降低培养时的温度并延长培养时间和适当提高摇床转速，确实可提高目的蛋白在上清中的表达，另外机械破碎法优于化学破碎法，溶菌酶的生物活性可能会影响目的蛋白的表达和完整性。 2.2 用12%SDS-PAGE鉴定亲和层析纯化的血管性血友病因子A1、血管性血友病因子G1324S纯度由于超声波破碎法诱导破碎的血管性血友病因子A1在上清中的表达优于溶菌酶破碎法，所有后续实验均采用超声波破碎法诱导表达和破碎，提取上清液。由于目的蛋白的N端均带有His标签，所以可以通过镍柱(HisTrapTM HP)亲和层析来对目的蛋白进行纯化。按材料与方法中所述的纯化方法将破碎后分离的上清液纯化，并将纯化后得到的目的蛋白进行12%SDS-PAGE检测，结果显示：①血管性血友病因子A1：与诱导前相比，诱导后菌体蛋白泳道于相对分子质量23 000附近新增一蛋白条带，与预计的目的蛋白相对分子质量一致，且纯化后在此位置出现单一蛋白条带。纯化后的目的蛋白主要存在于 300 mmol/L咪唑洗脱液部分(图2A)。②血管性血友病因子G1324S：与诱导前相比，诱导后的菌体蛋白泳道于相对分子质量31 000附近新增一蛋白条带，与预计的目的蛋白相对分子质量一致，且纯化后在此位置出现单一蛋白条带。纯化后的目的蛋白主要存在于200 mmol/L咪唑洗脱液部分(图2B)。说明血管性血友病因子A1和血管性血友病因子G1324S能够进行正确的表达，并且纯化出的目的蛋白具有较高的纯度。 2.3 Western-blot测定纯化产品的免疫活性血管性血友病因子A1与血管性血友病因子G1324S的N端都带有6×His标签，所以用兔IgG抗6×His多克隆抗体(一抗)和羊抗兔IgG HRP标记多克隆抗体(二抗)进行Western-blot鉴定，证实相对分子质量为23 000和31 000的蛋白条带分别为血管性血友病因子A1与血管性血友病因子G1324S(图3)，说明纯化的蛋白具有免疫学活性。 2.4 血管性血友病因子A1与血管性血友病因子G1324S的功能测定方差分析结果表明，底板功能化与流体剪应力均对血小板黏附数量产生显著影响，前者F=448.12，P= 1.22×10-15；后者F=89.60，P=5.86×10-8；且存在二因素的交互作用(P=2.24×10-6)。图4显示所纯化的蛋白A1及其突变体G1324S都具有黏附血小板的生物学功能，但通过图4A上下2组照片的比较，可明显看出血小板在G1324S上的黏附数要低于其在野生型A1的数目。图4B的实验数据表明，实验组(包括野生型A1和其突变体G1324S)黏附的细胞数要显著高于其实验对照组和空白对照组(P < 0.01)；而实验对照组又显著低于空白对照组(P < 0.01)，说明BSA能够有效阻断血小板与底板之间的物理黏附，但又不影响血小板与A1分子间的特异性相互作用，以保证实验组的细胞黏附确实是由于A1与血小板膜上的糖蛋白分子GPIbα特异相互作用所介导的。同时，对每个剪切应力下铺设不同A1分子的底板进行两两比较，发现：0.1 Pa组，野生型A1所黏附的细胞数比突变体G1324S组稍多，但差异无显著性意义(P=0.08)；1 Pa组，野生型A1上黏附的细胞数显著高于其突变体G1324S组(P < 0.01)，即随着流体剪应力从0.1 Pa提高1 Pa，G1324S突变组所黏附细胞数下降的幅度(46.8%)要远高于野生型组(20.5%)，说明突变后的A1分子的功能有所减弱，在高速流场中，其与GPIbα的结合亲和力更是大幅下降，完全不足以保证止血所必需的最低限度血小板的黏附。"

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