Alpha-thrombin is a highly specific serine protease generated by proteolytic activation of the zymogen prothrombin (1). During coagulation, thrombin cleaves fibrinogen to form fibrin, leading to the ultimate step in coagulation, the formation of a fibrin clot. Thrombin is also responsible for feedback activation of the procofactors factor V and factor VIII. Thrombin has also been reported to activate factor XIII and platelets, and also functions as a vasoconstrictor protein. The procoagulant activity of thrombin is arrested in two ways: 1) inhibition by either heparin cofactor II or the antithrombin III/heparin complex; or 2) complex formation with thrombomodulin. Formation of the thrombin/thrombomodulin complex results in the inability of thrombin to cleave fibrinogen and activate factors V and VIII, but increases the efficiency of thrombin for activation of the anticoagulant, protein C.
Thrombin is a two chain enzyme composed of an NH2-terminal "A" chain (Mr=6,000) and a COOH-terminal "B" chain (Mr=31,000) which remain covalently associated through a single disulfide bond. Human thrombin is 13 amino acids shorter than the bovine thrombin due to a thrombin cleavage site on the human protein that is not present in the bovine protein.
Thrombin is also utilized for site specific cleavage of fusion proteins expressed in bacteria (9-11). A thrombin sensitive site is incorporated between the recombinant protein of interest and peptides or proteins which facilitate purification and/or expression. The target protein is released from the expressed hybrid by cleavage with thrombin. Thrombin can then be easily removed by affinity chromatography.
Human, bovine and mouse thrombin are prepared from purified prothrombin using a modification of the Lundblad procedure (1) as described by Nesheim et al. (2). Thrombin is supplied in 50% (vol/vol) glycerol/H2O and should be stored at -20oC. Purity is determined by SDS-PAGE analysis and activity is measured in a thrombin specific clotting assay, and compared to standardized NIH thrombin. Thrombin is also available with the active site blocked with either DFP, FPRck, or biotinlyated FPRck.
Cleavage of Fusion ProteinsIn addition to its broad application in coagulation research thrombin can be used for site specific cleavage of fusion proteins. A thrombin sensitive site is incorporated between the recombinant protein of interest and peptides or proteins which facilitate purification and/or expression. The target protein is released from the expressed hybrid by cleavage with thrombin. Thrombin can then be easily removed by affinity chromatography. Lot to lot consistency ensures reproducible results every time. For experiments involving cell cultures, please contact us to discuss custom, low endotoxin lots designated for cell culture use.
Haemtech Biopharma可以运行多种分析技术，以提供对蛋白质药物产品或物质的生化分析，或帮助进行污染物鉴定。我们的测定，测试和技术菜单包括：应用领域IVIG药物的血栓形成性过程中杂质分析与止血有关的抗药物抗体测试（免疫原性）例如抗凝血因子抗体宿主细胞蛋白质的鉴定，定量和缓解在制品和在制品中药品的稳定性和释放测试与止血相关的重组蛋白的效力和纯度测定血浆蛋白分析蛋白质的结构表征翻译后修饰二硫键映射构象变化凝血测定试剂（例如凝血活酶）的表征定制的凝血产品检测应用药代动力学研究分析鉴定/验证工艺/产品验证蛋白质纯化方法的发展强迫降解研究方法高效液相色谱反相（RPC）大小排除（SEC）离子交换（IEC）疏水相互作用（HIC）金属亲和力（IMAC）蛋白质A（用于IVIG分析）SDS页面减少和不减少一维和二维考马斯亮蓝染色银染荧光染色免疫印迹多重分析定量化凝血测定因子分析（II，V，VII，VIII，IX，X，XI，XII，XIII等）PTPTPT行动计划福尔摩斯测试其他ELISA法各种显色测定因子分析（II，V，VII，VIII，IX，X，XI，XII，XIII等）其他翻译后修饰分析糖基化，硫酸化，磷酸化，羟基化，脱酰胺，乙酰化，氧化，氨基甲酸酯化和γ-羧化凝血酶生成测定对于IVIG和其他抗药物抗体ELISA（ADA）抗原料药抗体抗宿主细胞蛋白的抗体抗污染物或分解产物的抗体鉴定未知质谱N端测序残留水分测定顶空真空测量pH值渗透压折光率总蛋白质配方评估
