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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。
In the process of studying the structure-activity relationship of staphylokinase, it is found that staphylokinase easily forms dimers and even multimers, which is not conducive to the clinical application of staphylokinase. In order to explore the formation mechanism of polymer to develop a new type of staphylokinase which is not easily polymerized, based on the structure model of staphylokinase X-ray diffraction, the molecular docking software GRAMMV1033 was used to predict the expression of staphylokinase Possible binding sites for the mer. There are two main possible ways for the formation of the binding region, through strong hydrophobic interaction and hydrogen bonding. According to the model, a staphylokinase mutant RGD-Sk designed to reduce the polymerization capacity was designed. The substitution of F1 1 1 for D1 1 1 and K1 0 9 for R1 0 9 resulted in the formation of an RGD structure in the molecule, May have inhibition of platelet aggregation. The RGD-Sk gene was constructed by site-directed mutagenesis and DNA recombination technology and was expressed in E. coli prokaryotic expression system. RGD -Sak exists in the form of inclusion bodies. The inclusion body was washed, dissolved in 8mol / L urea, diluted and refolded, and separated by ion-exchange chromatography in one step. The purity of RGD-Sak was more than 95%. The molecular weight was in agreement with the theoretical value Activity 5 × 10 4 HU / mg. The Km and Kcat values of plasminogen catalyzed by the complexes formed by RGD-Sk and plasmin were 1 2 40 μmol / L and 0 81s-1, respectively. RGD -Sak shows very poor polymerization ability. This study laid the foundation for the development of novel staphylokinase molecules that prevent the formation of dimers.