重组抑肽酶的表达纯化和活性测定

Expression of Recombinant Aprotinin in E.coli and the Identification of Its Biological Activity

抑肽酶是一种用途广泛的丝氨酸蛋白酶抑制剂.用基因重组技术将化学合成的抑肽酶基因插入表达载体pET28a,并转化至大肠杆菌Rosetta(DE3)中,经IPTG诱导后,表达产物以包涵体形式存在于菌体内包涵体纯化后经复性及CM-阳离子交换层析后,从每升培养液可获得2.4 mg抑肽酶,纯度可达90%.活性测定结果显示,重组抑肽酶可竞争性抑制纤溶酶对小分子底物S2251的水解活性.抑制常数(Ki)为(0.6±0.3)nmol/L,与天然抑肽酶的抑制活性相似.

Aprotinin is a 58 amino acid bovine protease inhibitor which inhibits a range of serine proteases including trypsin, chymotrypsin, plasmin, kallikrein and trombin. This single-chain polypeptide with the molecular mass of 6. 5 kDa and the PI of 10.5 is crosslinked by three disulphide-bond bridges. It was initially isolated from bovine pancreas by Kunitz and Northrop. Aprotinin has been widely used in various fields. It can be used as inhibitor in protein purification procedure and tissue culture to prevent the degradation. In clinical use, because of its antifibrinolytic properties, aprotinin can be used in cardiac surgery to decrease postoperative blood loss and preserve platelet function during cardiopulmonary bypass. Due to the large requirement of aprotinin, the methods to produce aprotinin with high homogeneity and yields have been studied. Most of the reports regarding this topic are about purification of aprotinin from bovine tissues. But these purification procedures are complicated and costly. So the development of effective recombinant expression system is necessary. In this study, the deoxyoligonucleotide encoding aprotinin was synthesized according to the codon prefernce of E. coll. The DNA sequence was amplified by PCR and inserted into the expression vector pET28a between the NdeI and XhoI sites. The recombinant expression plasmid was then transformed into E. coli strain Rosetta （DE3）, and recombinant aprotinin （r-aprotinin） was expressed as inclusion body by IPTG induction. The amount of r-aprotinin expressed accounted for 8%-10% of the total bacterial proteirL After purification of inclusion body, renaturation and CM cation-exchange chromatography, 2.4 mg r-aprotinin was obtained from one liter of shaking flask culture of E. coli with homogeneity greater than 90%. Kinetic studies demonstrated that r-aprotinin competitively inhibited the amidolytic activity of plsamin against small substrate S2251. The inhibition constant（Ki） was （0.6±0.3） nmol/L, which was similar to that of the native aprotinin.