人组织激肽释放酶成熟蛋白的纯化及活性分析

Preparation in Small Scale, Purification and Bioactivity Analysis of Mature Human Tissue Kallikrein

目的 制备纯化人组织激肽释放酶 ,并检测其生物活性 ,为中试放大及纯化工艺奠定基础。方法 使用麦芽糖 (MBP)融合分泌载体pMBP P ,构建并筛选出 1株工程菌株 ,在 6L培养基中经IPIG诱导表达人组织激肽释放酶融合蛋白。目的蛋白经亲和层析纯化及凝血酶切割后 ,采用电位滴度法测定其活力。结果 筛选出的工程菌株表达相对分子质量约 70 0 0 0的激肽释放酶融合蛋白 ,大小与预计大小相符。纯化后共获得 2 8mg蛋白 ,并具有水解苯甲酰精胺酸乙酯 (BAEE)的能力。结论 成功进行了人组织激肽释放酶融合成熟蛋白的小量制备 ,经纯化后的产物具有生物活性 ,为中试放大、纯化工艺研究奠定了基础。

Objective To prepare and purify mature human tissue kallikrein by using E.coli system and analyze the bioactivity of the purified protein.Methods Insert the gene fragment encoding mature human tissue kallikrein into plasmid pMBP-P and transform to E.coli TOP 10F' strain.Inoculate the recombinant strain into a 6L medium for expression of human tissue kallikerein fusion protein under induction of IPTG.The expressed protein was purified by affinity chromatography,digested with thrombin,and analyzed for bioactivity by potentiometric method.Results The human tissue kallikrein fusion protein with a relative molecular weight of about 70 000,which was consistent with the expected,was expressed. After purification,25 mg of protein was harvested. Potentionmetric method showed the hydrolysis of BAEE with the expressed protein.Conclusion Mature human tissue kallikrein with bioactivity was successfully prepared in small scale and purified. It laid a foundation of study on the procedure of pilot and large-scale preparations of the protein.