摘要
以融合蛋白的形式，在Ｅ．ｃｏｌｉ中经温度诱导表达了小Ｃ肽人胰岛素原类似物（Ｂ－Ｒ２－Ａ）．表达的融合蛋白可占细胞总蛋白６８％．经磺酸解，及初步分离Ｓ－磺酸型融合蛋白，再经ＣＮＢｒ裂解后，进行还原重组，ＨＰＬＣ分离纯化等步骤，每升发酵液可得到Ｂ－Ｒ２－Ａ约５０ｍｇ。经酶促转化及ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－２５纯化，得重组人胰岛素约２０ｍｇ，其氨基酸组成与人胰岛素相同，并具有与猪胰岛素相同的生物活性．
The fused“Mini-C”human proinsulin analog (B-R2-A) was expressed in E. coli, induced by temperature.The expression level accounted for 68% of total bacterial proteins.The fused human B-R2-A was converted to crude fused B-R2-A S-sulfonate using oxidative sulfitolysis, isolated by Sephadex G-75 gel filtraction,and then was cleaved by CNBr.After reduction,reconstitution, and isolation with DEAE-5pw HPLC,50mg B-R2-A could be obtained (from 1 liter of fermentation culture).The B-R2-A then converted to human insulin by a combination cleavage with trypsin and carboxypiptidase B.The total yield of human insulin was about 20mg/L cell culture.The amino acid composition of the recombinant human insulin are in agreement with human insulin and the biological activity is 32.5IU/mg as judged by mouse blood sugar assay.To whom correspondence should be adressed.
关键词
小C肽
胰岛素原类似物
胰岛素
基因工程
Mini C”human proinsulin analog
E.coli
Human insulin
Isolation and purification
