By using PCR and DNA recombination,two fusion genes of humanized mouse anti human fibrin scFv and low molecular weight single chain urokinase (Scu PA 32K)was constructed.The difference of these two fusion genes lay in...By using PCR and DNA recombination,two fusion genes of humanized mouse anti human fibrin scFv and low molecular weight single chain urokinase (Scu PA 32K)was constructed.The difference of these two fusion genes lay in the linker between two moieties,one was (Ala) 3 and another was (Gly 4Ser) 3.These two fusion genes were both overexpressed in E.coli with the expression level at 30%.Both expression products showed the activity of binding antigen D Dimer and activating plasminogen after the denaturation and renaturation,but under general refolding conditions,the one with linker (Gly 4Ser) 3 showed better effect in the renaturation of fusion protein.展开更多
A 150 -156 amino acids-deleted single-chain urokinase-type plasminogen activator (dscu-PA) and its rccombinant wild-type counterpart (rscu-PA) were both expressed in Escherichia coli. After denaturation and renaturati...A 150 -156 amino acids-deleted single-chain urokinase-type plasminogen activator (dscu-PA) and its rccombinant wild-type counterpart (rscu-PA) were both expressed in Escherichia coli. After denaturation and renaturation in nitro, the expressed products were both purified lo a single silver-stained band by means of IgG affinity chromatography. After activation by plasrran, similar enzymatic constants based on the hydrolysis of synthetic substrate S2444 by the two-chain molecular forms of dscu-PA and rscu-PA, or native tcu-PA were observed, suggesting that no impairment had been exerted on the catalytic active site of dtcu-PA by the 150-156 amino acids deletion. In both in vitro fibrin-clot and 125I-fibrin sepharose lysis tests, dtcu-PA showed a significantly higher fibrinolytic activity than rtcu-PA or rscu-PA. Hardly any effect on the concentration of fibrinogen in plasma was found in dtcu-PA. It was concluded that dtcu-PA had a higher fibrin specificity and that tcu-PA could be provided with better fibrin specificity by means of mutation.展开更多
文摘By using PCR and DNA recombination,two fusion genes of humanized mouse anti human fibrin scFv and low molecular weight single chain urokinase (Scu PA 32K)was constructed.The difference of these two fusion genes lay in the linker between two moieties,one was (Ala) 3 and another was (Gly 4Ser) 3.These two fusion genes were both overexpressed in E.coli with the expression level at 30%.Both expression products showed the activity of binding antigen D Dimer and activating plasminogen after the denaturation and renaturation,but under general refolding conditions,the one with linker (Gly 4Ser) 3 showed better effect in the renaturation of fusion protein.
基金Project supported by the National High Technology Development Program of China.
文摘A 150 -156 amino acids-deleted single-chain urokinase-type plasminogen activator (dscu-PA) and its rccombinant wild-type counterpart (rscu-PA) were both expressed in Escherichia coli. After denaturation and renaturation in nitro, the expressed products were both purified lo a single silver-stained band by means of IgG affinity chromatography. After activation by plasrran, similar enzymatic constants based on the hydrolysis of synthetic substrate S2444 by the two-chain molecular forms of dscu-PA and rscu-PA, or native tcu-PA were observed, suggesting that no impairment had been exerted on the catalytic active site of dtcu-PA by the 150-156 amino acids deletion. In both in vitro fibrin-clot and 125I-fibrin sepharose lysis tests, dtcu-PA showed a significantly higher fibrinolytic activity than rtcu-PA or rscu-PA. Hardly any effect on the concentration of fibrinogen in plasma was found in dtcu-PA. It was concluded that dtcu-PA had a higher fibrin specificity and that tcu-PA could be provided with better fibrin specificity by means of mutation.
