重组猪胰蛋白酶突变体基因在大肠杆菌中的克隆、表达与活性分析  

Cloning and Expression of Porcine Trypsin Mutant Gene in Escherichia coli and Its Activity Analysis

作　　者：杨敏[1,2] 黄潇 张一唯 平宪卿 龙军[3] 曹荣月[1] 

机构地区：[1]中国药科大学,江苏南京210009 [2]江苏未名生物医药有限公司,江苏常州213000 [3]南京中医药大学,江苏南京210046

出　　处：《药物生物技术》2015年第4期283-287,共5页Pharmaceutical Biotechnology

基　　金：国家级大学生创新创业训练计划项目(No.J1030830);江苏高校优势学科建设工程资助项目(PAPD);mi R-26a靶向TET酶在胰腺成体干细胞分化过程中的作用及机制研究(中央高校基金杰出人才引导项目);国家自然科学基金(No.81172973;No.81373232)

摘　　要：胰蛋白酶是肽链内切酶,专一性水解多肽链中赖氨酸和精氨酸羧基端形成的肽键。胰蛋白酶切割胰岛素原生成胰岛素,在胰岛素的生产中发挥着重要作用,但由于胰蛋白酶对赖氨酸和精氨酸的切割选择性差别不大,会在切割反应中产生副产物。所以研究开发对底物切割选择性增强的胰蛋白酶,减少胰岛素生产过程中副产物的产生具有较高的应用价值。以猪源胰蛋白酶原基因为模板,通过重叠延伸产生特异位点突变的方法扩增突变胰蛋白酶原(Tg M)基因,该基因编码猪胰蛋白酶的Ser172Ala突变体。将Tg M基因插入到表达载体p ET28a中,转化E.coli DH5α,利用卡那霉素抗性筛选出构建成功的重组质粒p ET28a-Tg M并将其转化E.coli BL21(DE3),构建含Tg M基因的工程菌株并诱导表达。14%SDS-PAGE显示,重组菌经乳糖诱导后表达了目的蛋白,并以包涵体形式存在。包涵体经尿素裂解液变性后,通过稀释法复性,复性液超滤处理并用胰蛋白酶切割,经DEAE-FF离子交换层析分离纯化。纯化蛋白经SDS-PAGE分析,结果表明与胰蛋白酶条带位置相同;用紫外分光光度法检测酶活力,测得突变胰蛋白酶的酶活力为1 300 U/m L,比活力为1 566.27 U/mg。将最终纯化获得的突变胰蛋白酶用于胰岛素原的切割,HPLC检测分析结果表明,突变胰蛋白酶对底物中Arg的切割选择性大于Lys,因此在对胰岛素原的切割反应中,副产物的量明显减少。Trypsin is a kind of endopeptidase with known activity as hydrolyzing polypeptide chain on lysine and arginine carboxyl end with this activity trypsin could cut proinsulin to produce insulin. So it plays an important role in the insulin industrial production.Because of the low selectivity for lysine and arginine,there are always a lot of byproducts associated with the generation of insulin. So enhancing the substrate selectivity of trypsin to reduce the by-products has a very high application value. The trypsinogen gene mutation（ Tg M） encoding Ser172 Ala trypsinogen was amplified by overlap extension polymerase chain reaction and inserted into expression plasmid p ET-28 a. The recombinant plasmid was transformed into Escherichia coli DH5α first. Clones which contain p ET28a-Tg M were selected by growth on kanamycin plates. Then the recombinant plasmid p ET28a-Tg M was transformed into Escherichia coli BL21（ DE3） to be induced to express. From the result of the 14% SDS-PAGE,the recombinant plasmid could express the target protein by lactose induction and the target protein has accumulated intracellularly in the form of inclusion bodies. The pellet was resuspended in denaturing solution and renatured by the dilution method. The refolding solution with proteins was deat with ultrafiltration. Samples were treated with standard trypsin after ultrafiltration and purified with DEAE-FF. After purification,target protein and the standard trypsin were in the same position showed by the 14% SDS-PAGE. The enzyme activity was 1 300 U / m L and the specific activity was1 566. 27 U / mg. Purified target protein was used to convert the pro-insulin into insulin. The product was assayed by HPLC which showed that the amount of conversion by-product is dramatically reduced compared with standard trypsin. In conclusion,the trypsin mutant has a pronounced selectivity for arginine residues.

关 键 词：胰蛋白酶突变体 蛋白表达 包涵体 蛋白复性 胰岛素 离子交换层析 

分 类 号：R576[医药卫生—消化系统]