人胰岛素原类似物(BKRA)基因的合成与表达

Synthesis of Human Proinsulin Analog(BKRA) Gene and Its Expression in E.coli

为了利用基因工程生产胰岛素，按照已知的人胰岛素Ａ、Ｂ链氨基酸序列和大肠杆菌偏爱的氨基酸密码子设计并合成了人胰岛素原类似物（ＢＫＲＡ）基因，其中以赖（Ｋ）－精（Ｒ）二肽编码区取代人胰岛素原Ｃ肽编码区．为了避免其编码蛋白在大肠杆菌中表达时被降解，通过人工接头将２个ＢＫＲＡ基因串联起来，接头部分氨基酸序列为Ａｒｇ－Ａｒｇ－Ａｓｎ－Ｓｅｒ．将串联的ＢＫＲＡ基因克隆到表达载体ｐＥＴ－２８ａ（＋），实现了在大肠杆菌中的融合表达，表达产物以包含体形式存在，约占细菌总蛋白２４％．表达产物氨基末端具有六组氨酸肽段，以ＨｉＴｒａｐ凝胶进行亲和层析，一步纯化可达纯度９５％以上．放射免疫测定表明，纯化的融合蛋白具有胰岛素抗原活性．

Human proinsulin consists of three peptide segments,chain A and chain B linked by chain C(or peptide C).The normal chain C is 35 amino acids long.Study showed that chains A and B contained sufficient structural information to form the native insulin and that the proinsulin analog with a “mini C” as short as two amino acids could be processed into insulin.For the production of recombinant human insulin,a human proinsulin analog (BKRA) gene with E.coli biased amino acid codons was synthesized according to the known amino acid suquence of human insulin.In the BKRA gene,a lysine(K) arginine(R) dipeptide coding region was designed to replace the coding region of peptide C of natural human proinsulin.To avoid the small BKRA protein to be degraded while expressing in E.coli ,a synthetic linker sequence was used to join tandemly two BKRA genes.The connective amino acid sequence between two BKRAs would be Arg Arg Asn Ser.When the expressed proteins were treated with cyanogen bromide (CNBr),two molecules of BKRA would be produced.In the next steps of proteinase digestion the connector tetrapeptide and the “mini C” dipeptide would be removed simultaneously,producing human insulin.The tandemly linked BKRA gene was cloned into pET 28a(+),an expression plasmid,and expressed in E.coli in fusion protein fashion.The proteins were aggregated into the inclusion body,and weighed about 24% of the total proteins of the expression bacteria.The expressed proteins contained a six histidine peptide in the amino terminus,which were easily purified by HiTrap affinity matrix,one step of capture could get purity of 95%.Human insulin antigenicity of the purified fusion protein was detected by radioimmunoassay.The results showed that a high level expression recombinant bacteria of human proinsulin analog was constructed.