一种新型葡激酶分子的设计基因构建表达纯化及性质研究

Rational Design,Over-expression and Characterization of a Novel Staphylokinase with Lower Tendency of Polymerization

在葡激酶构效关系的研究过程中发现 ,葡激酶易形成二聚体 ,甚至多聚体 ,不利于葡激酶的临床应用。为了探究聚合体的形成机制以研制不易聚合的新型葡激酶分子 ,在葡激酶X射线晶体衍射结构模型的基础上 ,采用分子对接软件GRAMMV1 0 3,以高分辨率整体对接方式预测了葡激酶二聚体可能的结合区。结合区主要有两种可能的形成方式 ,通过强疏水相互作用及氢键结合。根据模型设计了旨在降低聚合能力的葡激酶突变体RGD -Sak ,将F1 1 1置换为D1 1 1 ,并且改变K1 0 9为R1 0 9,恰好使分子中形成RGD结构 ,使新型分子还可能具有抑制血小板聚集作用。利用定点突变及DNA重组技术 ,构建了RGD -Sak基因 ,并利用大肠杆菌原核表达系统进行了高效表达。RGD -Sak以包涵体形式存在 ,包涵体经洗涤 ,8mol/L尿素溶解 ,稀释复性 ,离子交换色谱一步分离得到电泳纯的RGD -Sak ,纯度达 95%以上 ,分子量与理论值相符 ,比活性 5× 1 0 4 HU/mg。RGD -Sak与纤溶酶形成的复合物催化纤溶酶原的Km、Kcat值分别为 1 2 40 μmol/L、 0 81s- 1 。RGD -Sak显示了很弱的聚合能力。此研究为研制防止二聚体形成的新型葡激酶分子打下了基础。

Staphylokinase has strong tendency to polymerize,which may hamper its clinical use.On the basis of crystal structure of staphylokinase,the dimer structure was modeled by software GRAMM V1 03 with high resolution generic docking parameters.Two kinds of binding interfaces were generated,mainly linked by hydrophobic interaction and hydrogen bonds.The computer model of staphylokinase dimer provides a template for the design of improved staphylokinase without tendency to form polymer.A novel variant of staphylokinase(RGD Sak)was designed based on the model.RGD Sak gene was constructed by substitution mutagenesis of Lys 109 and Phe 111 with Arg 109 and Asp 111 respectively,confirmed by nucleotide sequencing.The mutant cDNA was ligated with prokaryotic expression vector pLY 4 and transformed into E.Coli JF1125.After temperature induction,over 50% expression level of RGD Sak was achieved.RGD Sak was isolated and purified by washing and solubilization of inclusion body,renaturation and ion exchange chromatography.The final product displayed a single band with a corresponding molecular weight of 15 5 kD in non reducing SDS PAGE with 95% of purity and 5×10 4 HU/mg of specific activity.The K M、K cat value for the activation of plasminogen with RGD Sak was 12 40 μmol L -1 、0 81 s -1 ,respectively.RGD Sak show lower tendency to polymerize after incubated with 0 9% NaCl.This study provided the basis for further reconstruction of Sak by protein engineering. [