人纤溶酶原K5突变体在大肠杆菌中的优化表达  

Optimal Expression and Activity Detection of Human Plasminogen Mutant Kringle 5 in E.coli

作　　者：李朝阳 温南[2] 杨中汉[2] 蔡卫斌[2] 李明友 刘祖国 高国全[2] 杨霞[2] 

机构地区：[1]眼科学国家重点实验室//山大学中山眼科中心,广东广州510060 [2]中山大学中山医学院生物化学教研室,广东广州510080 [3]广州市启源生物科技有限公司,广东广州510630

出　　处：《中山大学学报（医学科学版）》2008年第2期221-225,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金资助项目(30570372;30600724;30700120);中华医学基金会资助项目(CMB-SUMS学者项目98-677);教育部新世纪人才计划(NCET-04-0792);广东省自然科学基金研究团队项目(06201946);广东省科技计划项目重大专项(2005A10902003;2006B35502001);广州市科技攻关重点基金资助项目(2006Z3-E4111;2007Z3-E5041)

摘　　要：【目的】提高K5突变体(mK5)在原核系统的单位表达量。【方法】调整可能影响表达的参数:抗生素浓度、pH、A600值、IPTG终浓度、诱导时间、诱导温度,同时固定其它参数。通过电泳分析,获得mK5在原核系统pET22b-BL21的最优表达条件;组氨酸结合柱亲和层析、纯化获得mK5蛋白,SDS-PAGE和Western-blot方法鉴定mK5蛋白;MTT法分析mK5对人视网膜毛细血管内皮细胞(HRCEC)增殖的影响。【结果】优化后的表达条件为:最适细菌培养温度为37℃,最佳抗生素浓度为50μg/mL羧苄青霉素,最适pH值为7.0,在A600为0.9～1.0时加入终浓度为1.0mmol/L的IPTG为最佳诱导条件,37℃诱导10h为最佳诱导时间;优化条件后mK5纯化蛋白得率为21mg/L,产量较优化前(15mg/L)提高近30%;mK5特异性地抑制HRCEC的增殖,IC50=40nmol/L。【结论】本研究确定了mK5蛋白诱导表达的最适参数,并获得具有生物活性的高纯度、高表达量的重组蛋白,为工业化大规模发酵生产提供了基本参数。[Objective ] To optimize and increase the expression of per unit of mutant K5 （mK5） in the prokaryotic expression system pET22b-BL21. [Methods] All kinds of factors which possibly effect the expression of prokaryotic system was optimized on the basis of process preexisting, such as： the antibiotic concentration, pH, A600, the final concentration of IPTG, the temperature and period of inducement etc. mK5 was purified by Ni^2＋-His bind resin affinity chromatography. The purity and identity of mK5 was examined by SDS-PAGE and Western blot analysis. The anti-proliferative effects of mK5 on primary human retinal capillary endothelial cell （HRCEC） was examined by MTT assay. [ Results ] The optimal parameter of expressing mK5 in the prokaryotic expression system pET22b-BL21 is： culture the E.coli with the recombinant in 37 ℃ pH 7.0 LB culture medium with carbenicillin 50 μg/mL by using the horizontal swing bed 230 r/min. While A600 goes to 0.9-1.0, adding IPTG to final concentration 1.0 mmol/L, collects the bacterial precipitation by centrifugation after the E.coli has been induced for 10 hours in 37℃. An average of 21 mg of purified mK5 was obtained from the periplasmic fraction of 1 liter of culture, which increase 30% than before （15 mg/L）, mK5 inhibited proliferation of primary endothelial cells in dose-dependent manner, IC50 is 40 nmol/L. [Conclusion] The optimal parameter of expressing mK5 in the prokaryotic expression system was identified and high yield active mK5 was obtained in the present study. These data can be used in the large scale industrial zymosis of the recombination protein mK5.

关 键 词：人纤溶酶原K5 缺失突变体 血管新生 优化表达 

分 类 号：Q784[生物学—分子生物学]