重组鲨鱼血管生成抑制因子功能活性区蛋白纯化工艺的优化

Optimization of purification procedure for activity domain of recombinant shark angiogenesis inhibition factor

目的优化重组鲨鱼血管生成抑制因子(Shark angiogenesis inhibition factor,SAIF)功能活性区(aSAIF)蛋白的纯化工艺。方法利用离子交换层析和镍柱亲和层析对带有组氨酸标签的融合蛋白His6-SUMO-aSAIF进行纯化。纯化后的融合蛋白经SUMO蛋白酶切除小分子泛素样修饰蛋白(Small ubiquitin-related modifier,SUMO)标鉴,再经镍柱二次亲和层析获得非融合aSAIF蛋白,采用WST-8法对其进行血管生成抑制活性检测。结果优化的纯化工艺参数为:离子交换层析采用0.2 mol/L的NaCl进行洗脱;亲和层析的洗脱液为20 mmol/L磷酸盐,500 mmol/L NaCl,250 mmol/L咪唑,pH 7.4;SUMO蛋白酶与融合蛋白的最佳酶切比例为1∶480,酶切时间为1 h。最终获得的aSAIF蛋白纯度可达95%,具有较好的抑制血管内皮细胞增殖的活性,且呈剂量依赖性。结论已成功建立了aSAIF的纯化工艺,为后续大规模生产及抑制血管生成机制的研究奠定了基础。

Objective To optimize the purification procedure for activity domain of recombinant shark angiogenesis inhibition factor SAIF）,i.e.aSAIF.Methods Fusion protein His6-SUMO-aSAIF with His tag was purified by ion exchange chromatography and nickel ion affinity chromatography,in which the SUMO（small ubiquitin-related modifier） tag was digested with SUMO protease,then further purified by nickel ion affinity chromatography to obtain non-fusion aSAIF which was determined for angiogenesis-inhibiting activity by WST-8 method.Results The main parameters of purification procedure were optimized as follows： ion exchange chromatographic column was eluted with 0.2 mol / L sodium chloride;affinity chromatographic column was eluted with 20 mmol / L phosphate,500 mmol / L sodium chloride and 250 mmol / L imidazole at pH 7.4.The optimal ratio of SUMO protease to fusion protein was 1 ∶ 480,and the time for digestion was 1 h.The obtained aSAIF reached a purity of 95% and showed dose-dependent activity in inhibiting the proliferation of vascular endothelial cells.Conclusion The purification procedure for aSAIF was successfully developed,which laid a foundation of further large-scale production of aSAIF and study on mechanism of inhibiting angiogenesis.