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用限制性内切酶将人尿激酶原cDNA酶切成一系列基因片段 ,并分别在大肠杆菌中表达。发现其中 1个富含大肠杆菌稀有密码子AGG(精氨酸 )的片段表达量低 ,成为人尿激酶原cD NA在E .coli中高效表达的限制因素。将富含AGG(精氨酸 )的片段在E .coliBL2 1 CodonPlusTM RIL中表达 ,通过该菌株引入dnaY基因 (即tRNAagg/aga(Arg) )使该片段的表达量提高了 10倍。最后用同样的方法提高全长人尿激酶原cDNA在大肠杆菌中的表达量 ,使其表达量达到全菌蛋白的5%。
Human urokinase cDNA was digested with restriction enzymes into a series of gene fragments and separately expressed in E. coli. It was found that one of the fragments rich in Escherichia coli rare codon AGG (arginine) was low expression, as human urokinase cD NA efficient expression in E.coli limiting factor. The AGG (arginine) -rich fragment was expressed in E. coli BL21 CodonPlusTM RIL, and the introduction of the dnaY gene (ie, tRNAagg / aga (Arg)) by this strain resulted in a tenfold increase in the expression of this fragment. Finally, in the same way, the expression of full-length human urokinase cDNA in E. coli was increased to reach 5% of the total bacterial protein.