重组葡激酶的基因克隆与序列测定

从烧伤病房病人烧伤感染创面分离到 9株金黄色葡萄球菌 ,经纤溶活性测定 ,选纤溶活性最高的作为 PCR扩增模板。将扩增出的葡激酶基因片段插入 p UC1 8载体质粒 ,转化大肠杆菌 DH5 α,筛选出 PUC- SAK阳性克隆 ,经 DNA序列测定证实该基因片段为一种新的 DNA序列     

新型溶栓剂葡萄球菌激酶的研究进展

本综述从葡激酶的溶栓作用机制,包括与纤溶酶(原)等因子的结合与作用,葡激酶的高级结构,抗原性问题等方面概括了近年来有关葡激酶作为亲一代溶栓剂的研究成果,并指出进一步利用蛋白质工程,对葡激酶进行分子改造的设想。     

新型溶栓剂葡萄球菌激酶的研究进展

本综述从葡激酶的溶栓作用机制,包括与纤溶酶（原）等因子的结合与作用,葡激酶的高级结构,抗原性问题等方面概括了近年来有关葡激酶作为亲一代溶栓剂的研究成果,并指出进一步利用蛋白质工程,对葡激酶进行分子改造的设想。     

葡萄球菌激酶作为新型溶栓剂的研究进展

近年来对溶栓剂的研究取得了很好的成果,主要集中在单链尿激酶型纤溶酶原激活剂(scuPA),组织型纤溶酶原激活剂(tPA),葡萄球菌激酶(SaK)等。葡萄球菌激酶(SaK)是一种激活溶纤维蛋白的制剂,它与纤溶酶原(Plg)形成1∶1的复合体,使后者转变为纤溶酶(Pli)后激活其他分子变为Pli。从葡激酶的溶栓作用机制,包括与纤溶酶(原)等因子的结合作用,葡激酶的高级结构,抗原性等问题以及近年来有关葡激酶作为新一代溶栓的研究进展进行了综述,并指出进一步利用蛋白质工程,对葡激酶进行分子改造的设想。     

药用蛋白质的化学修饰

化学修饰是改善药用蛋白质的免疫原性,半衰期等生物学特性的有效方式,已经成为生物化学和分子生物学领域的研究热点之一。本文主要介绍常用化学修饰剂的种类及其性质,评述化学修饰的方式和原理,列举化学修饰蛋白质在生物医药领域的应用。     

葡激酶N端突变体的分子设计,高效表达与分离纯化

同源模建人微小纤溶酶原（ｍｉｃｒｏｐｌａｓｍｉｎｏｇｅｎ ,ｍｐｌｇ）的三维结构,建立葡激酶（ｓｔａｐｈｙｌｏｋｉｎａｓｅ,Ｓａｋ）,ｍｐｌｇ计算机分子对接的结构模型,模型与已有的实验结果基本相符。为研究葡激酶Ｎ端结构与功能的关系,进一步改造葡激酶分子,根据复合物结构模型设计了Ｎ端缺失１５个氨基酸的葡激酶突变体。     

重组葡激酶的高效表达及分离纯化

利用基因重组技术,将葡激酶基因克隆到枯草芽孢杆菌（B.subtilis)中,经过发酵培养,葡激酶被高效表达,并以可溶性活性状态分泌到胞外,本文报道利用SP Sepherose和S－200 Sephacryl二步柱层析来纯化葡激酶,最终纯度在98％以上,得率为50％－60％。     

RGD-葡激酶的凝胶过滤层析法复性及其纯化

构建的溶栓和抗栓双重功能的RGD-葡激酶突变体(RGD-Sak)在大肠杆菌中高表达,目的蛋白质以包涵体形式存在。为获得有活性的蛋白质,需要对包涵体进行变复性。利用凝胶层析方法对包涵体中RGD-Sak进行复性,并与稀释复性法进行比较,发现凝胶柱复性方法具有操作周期短、简便、成本低而高效等优点。复性后蛋白质用Q-Sepharose FF离子交换进一步纯化,纯度达95%,酪蛋白凝胶板活性测定表明两种复性法得到的蛋白质比活性相当。圆二色谱测定显示两种复性法得到的蛋白质的二级结构成份和谱形一致,说明在两种复性过程中完成了RGD-Sak分子的正确折叠。     

酶分子化学修饰研究进展

酶是高效生物催化剂,在工业和临床医药上应用广泛。但由于酶是蛋白质,稳定性差,且在生物体内有较强的免疫原性,因而严重制约了其应用。对酶分子进行化学修饰是提高其稳定性和降低免疫原性的有效途径。简要介绍几种改进酶催化特性的方法、酶分子修饰效果的分析与评价、酶通过化学修饰获得的新性质及其原理、酶化学修饰的研究动态等。     

重组葡激酶工程菌的构建方法

目的：利用N端缺失10个氨基酸的葡激酶（recombinant staphylokinase,rSaK）重组质粒,构建了表达可溶性rSaK126蛋白的工程菌,并研究不同条件下工程菌诱导表达目的蛋白含量的差异及纯化途径。 方法：采用细菌活化和培养方法诱导目的蛋白,并用SDS-PAGE测其含量,应用层析技术纯化蛋白。 结果：成功构建表达重组葡激酶的工程菌,表达的重组葡激酶蛋白约占菌体总蛋白的50％,经纯化后回收率为60%,纯度达99%以上。结论：成功构建高效表达重组葡激酶的工程菌,并获得了高含量、高纯度的目的蛋白。     

Construction and characterization of a novel staphylokinase variant with thrombin-inhibitory activity

Staphylokinase (SAK) is an effective thrombolysis agent for therapy of myocardial infarction. We have constructed a fusion SAK variant (SAK-HV) with a thrombin-binding domain composed of 12 amino acids from hirudin and expressed it in Escherichia coli and purified the resultant protein. SAK-HV maintained fibrinolytic activity similar to SAK and had anticoagulant activity attributable to its hirudin segment. Measurement of thrombin-binding activity in vitro demonstrated that SAK-HV possessed binding activity with thrombin while SAK did not. SAK-HV might thus be a more potent thrombolytic agent with anticoagulation property than SAK.     

Crystal structure of a staphylokinase: variant a model for reduced antigenicity.

Staphylokinase (SAK) is a 15.5-kDa protein from Staphylococcus aureus that activates plasminogen by forming a 1 : 1 complex with plasmin. Recombinant SAK has been shown in clinical trials to induce fibrin-specific clot lysis in patients with acute myocardial infarction. However, SAK elicits high titers of neutralizing antibodies. Biochemical and protein engineering studies have demonstrated the feasibility of generating SAK variants with reduced antigenicity yet intact thrombolytic potency. Here, we present X-ray crystallographic evidence that the SAK(S41G) mutant may assume a dimeric structure. This dimer model, at 2.3-A resolution, could explain a major antigenic epitope (residues A72-F76 and residues K135-K136) located in the vicinity of the dimer interface as identified by phage-display. These results suggest that SAK antigenicity may be reduced by eliminating dimer formation. We propose several potential mutation sites at the dimer interface that may further reduce the antigenicity of SAK.     

Staphylokinase as a Plasminogen Activator Component in Recombinant Fusion Proteins

The plasminogen activator staphylokinase (SAK) is a promising thrombolytic agent for treatment of myocardial infarction. It can specifically stimulate the thrombolysis of both erythrocyte-rich and platelet-rich clots. However, SAK lacks fibrin-binding and thrombin inhibitor activities, two functions which would supplement and potentially improve its thrombolytic potency. Creating a recombinant fusion protein is one approach for combining protein domains with complementary functions. To evaluate SAK for use in a translational fusion protein, both N- and C-terminal fusions to SAK were constructed by using hirudin as a fusion partner. Recombinant fusion proteins were secreted from Bacillus subtilis and purified from culture supernatants. The rate of plasminogen activation by SAK was not altered by the presence of an additional N- or C-terminal protein sequence. However, cleavage at N-terminal lysines within SAK rendered the N-terminal fusion unstable in the presence of plasmin. The results of site-directed mutagenesis of lysine 10 and lysine 11 in SAK suggested that a plasmin-resistant variant cannot be created without interfering with the plasmin processing necessary for activation of SAK. Although putative plasmin cleavage sites are located at the C-terminal end of SAK at lysine 135 and lysine 136, these sites were resistant to plasmin cleavage in vitro. Therefore, C-terminal fusions represent stable configurations for developing improved thrombolytic agents based on SAK as the plasminogen activator component.     

具有抗血小板聚集功能的新型葡激酶突变体的表达、纯化及性质

[目的]为解决溶栓后再栓塞问题,构建N-端含RGD(Arg-Gly-Asp)序列的葡激酶双功能突变体.研究突变体的表达和纯化,并进行性质分析.[方法]将突变后的葡激酶突变体序列连入pBV220质粒,转化大肠杆菌BL21进行表达.阳离子交换、凝胶过滤和阴离子交换三步层析法纯化表达产物,采用溶圈法对纯化产物进行生物学活性测定,并测定纯化产物对血小板聚集的抑制效应.[结果]PAGE扫描结果显示,葡激酶突变体蛋白在大肠杆菌BL21中的表达量约占菌体蛋白总量的40%～50%;三步层析纯化后,HPLC测定其纯度可达95%.酪蛋白凝胶板溶圈法测得其比活性分别为10.8×104和11.0×104HU/mg,与野生型葡激酶活性相当;且具有明显的抗血小板聚集活性,血小板聚集仪测定其血小板聚集抑制率分别为10.72%和19.71%,明显高于野生型葡激酶血小板聚集抑制率.本实验利用pBV220载体高效表达了葡激酶突变体基因,得到了高纯度、高活性的突变体蛋白,为葡激酶生产产业化和临床应用奠定了良好的基础.     

Kinetic and stoichiometric analysis of the modification process for N-terminal PEGylation of staphylokinase

Staphylokinase (SAK) is a therapeutic protein with promise for thrombolytic therapy of acute myocardial infarction. In this study, polyethylene glycol (PEG) aldehyde was used for N-terminal PEGylation of SAK to improve the pharmacological profiles of SAK. Due to the presence of the competitive PEGylation between the N terminus and the Lys residues, kinetic and stoichiometric analysis was carried out to investigate the process for the N-terminal PEGylation of SAK. To achieve this objective, size exclusion chromatography and tryptic peptide mapping were used to measure the PEGylation extent of SAK molecule and its specific amino acid residues, respectively.     

Novel self-cleavage activity of Staphylokinase fusion proteins: An interesting finding and its possible applications

Staphylokinase (SAK) is reported to have a serine protease domain with no proteolytic activity unlike other plasminogen activators like tissue plasminogen activator (t-PA) and urokinase. A unique protease property of Staphylokinase was observed when SAK was expressed as a fusion protein in inducible Escherichia coli expression vectors. This finding was further investigated by cloning and expressing different SAK fusions, both native and N-terminal deletions, with fusion tags like glutathione S-transferase (GST) and signal sequence of SAK in bacterial system. While all the N-terminal SAK fusions were found to self-cleave in crude and purified preparations, the C-terminal SAK fusion was stable. The cleavage property of Staphylokinase fusion proteins, inhibited by reduced glutathione and PMSF, was independent of its thrombolytic activity and also independent on the type of host employed for its expression. The serine protease domain of the SAK gene possibly lies between 20th to 77th amino acid and serine 41 of this region appears critical for such a cleavage property.     

抗凝血蛋白质分子的研究现状

凝血酶抑制剂,纤溶酶原激活剂（t-Pa,u-Pa,SK和SAK等）,蚯蚓纤溶酶和蛇毒抗凝血蛋白等是血栓类疾病领域的研究重点,纤溶分子突变体的应用,纤维蛋白水解特异性的增加,溶栓效率的提高以及半衰期的延长等是溶栓研究的方向。     

溶栓剂研究的新进展

近年来对溶栓剂的研究取得了很好的成果 ,主要集中在单链尿激酶型纤溶酶原激活剂 (scu PA) ,组织型纤溶酶原激活剂 (t PA) ,葡萄球菌激酶 (SAK)等 ,对分子进行设计改造 ,保留它们高效的溶栓功能同时赋予新的功能。在对分子空间结构域和活性功能区分析的基础上 ,研制出更有效的、更安全的溶栓制剂具有广阔的发展前景。     

Fibrin‐targeted plasminogen activation by plasminogen activator,PadA, from Streptococcus dysgalactiae

Bacterial plasminogen activators differ from each other in their mechanism of plasminogen activation besides their host specificity. Three‐domain streptokinase (SK) and two‐domain PauA generate nonproteolytic active site center in their cognate partner plasminogen but their binary activator complexes are resistant to α2‐antiplasmin (a2AP) inhibition causing nonspecific plasminogen activation in plasma. In contrast, single‐domain plasminogen activator, staphylokinase (SAK), requires proteolytic cleavage of human plasminogen into plasmin for the active site generation, and this activator complex is inhibited by a2AP. The single‐domain plasminogen activator, PadA, from Streptococcus dysgalatiae, having close sequence and possible structure homology with SAK, was recently reported to activate bovine Pg in a nonproteolytic manner similar to SK. We report hereby that the binary activator complex of PadA with bovine plasminogen is inhibited by a2AP and PadA is recycled from this complex to catalyze the activation of plasminogen in the clot environment, where it is completely protected from a2AP inhibition. Catalytic efficiency of the activator complex formed by PadA and bovine plasminogen is amplified several folds in the presence of cyanogen bromide digested fibrinogen but not by intact fibrinogen indicating that PadA may be highly efficient at the fibrin surface. The present study, thus, demonstrates that PadA is a unique single‐domain plasminogen activator that activates bovine plasminogen in a fibrin‐targeted manner like SAK. The sequence optimization by PadA for acquiring the characteristics of both SK and SAK may be exploited for the development of efficient and fibrin‐specific plasminogen activators for thrombolytic therapy.     

High-level expression of non-glycosylated and active staphylokinase from <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (~1 g/l) of extracellular glycosylated rSAK (~18 kDa) with negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated and highly active rSAK (~15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous rSAK of >95% purity which suitable for future structural and functional studies.