摘要
利用PCR技术从Streptococucspyogenes的基因组DNA中扩增了链激酶的编码基因ska,并进行了序列分析 ,利用基因删除及定点位变技术获得了删除了C 末端 42个氨基酸残基编码区的突变链激酶基因skaΔC42 ,第 5 9位Lys残基突变为Glu的突变链激酶基因skaK5 9E以及删除C 末端 42个氨基酸且第 5 9位Lys残基突变为Glu的突变链激酶基因skaΔC42K5 9E ,将ska及其三种突变体分别克隆到表达载体pET 1 5b上 ,构建分别表达野生型链激酶 (SK)、C 末端缺失 42个氨基酸残基的突变体 (SKΔC42 )、第 5 9位Lys残基突变为Glu的突变体 (SKK5 9E)及C 末端缺失 42个氨基酸且第 5 9位Lys残基突变为Glu突变体 (SKΔC42K5 9E)的表达载体pSK ,pSKΔC42 ,pSK K5 9E ,pSKΔC42K5 9E ,分别转化E .coliBL2 1 (DE3) ,IPTG诱导后在大肠杆菌中实现了高效表达 ,经亲和层析、离子交换层析及分子筛层析 ,获得了rSK、rSKΔC42、rSKK5 9E及rSKΔC42K5 9E 。
ska,the gene encoding the streptokinase was amplified by PCR from Streptococucs pyogenes genomic DNA and sequenced.Through DNA deletion and point mutation tequeniques,three mutants of ska,which were skaΔC42,skaK59E and skaΔC42K59E,were got.skaΔC42 encoded a muted streptokinase o which C-terminal 42 amino acid residues were deleted,skaK59E encoded a muted streptokinase in which Lys59 was muted to Glu59,skaΔC42K59E encoded a muted streptokinase in which Lys59 was muted to Glu59 and C-terminal 42 amino acid residues were deleted.ska and it's three mutants were cloned into pET-15b to construct pSK,pSKΔC42,pSK-K59E and pSKΔC42K59E expressing wild-type SK,SKΔC42,SKK59E and SKΔC42K59E,repectively.The four expressing vectors were introduced into E.coli BL21(DE3),overexpression of the four protein were achieved by IPTG induction.The four recombinant proteins were purified by nickel resin affinity chromatography,ion-exchange chromatography on DEAE-Sephadex column and gel filtration of Superdex 200 column.Activity analysis indicated that the recombinant SK and its three mutants showed the same activities.;
出处
《中国生物工程杂志》
CAS
CSCD
2004年第8期82-87,共6页
China Biotechnology
基金
山东省教育厅科技计划资助项目 (J99E)
