重组鲑鱼降钙素前体多肽的制备及其性质研究  

Preparation and Characterization of Recombinant Salmon Pre-calcitonin Peptide

作　　者：刘深基[1] 陈松森[1] 狄旭[1] 熊安琪[1] 张友鸿[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所,北京100005

出　　处：《中国生物化学与分子生物学报》2000年第5期650-654,共5页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：硫氧还原蛋白的第 38位 Met突变为 Ala的 Trx- s CT- Gly基因在 E.coli BL2 1 ( DE3)中得到高效表达 .用 Thio Bind亲和树脂纯化表达的融合蛋白 .结果说明 38位的 Met突变为 Ala不影响融合蛋白与树脂的特异性结合 ,融合蛋白的纯度达 90 %以上 .在含有 3mol/L尿素的 70 %甲酸中 ,室温 48h,至少 80 %融合蛋白被溴化氰裂解开 .采用 CM-纤维素吸附 ,用稀盐酸解吸附 ,得到纯度为92 %的降钙素前体多肽 s CT- Gly.氨基酸的序列分析结构表明 ,重组 s CT- Gly的 N端 1 0个氨基酸与预期一致 .在强酸性条件下 ,没有发生氨基酸的脱酰胺反应 ,氨基酸组成分析与预期基本一致 .质谱法测定的分子量为 3492 ,毛细管电泳测定的等电点 p I为 6.46,大鼠降钙素比活性为 1 90 IU/mg左右 ,与天然的人降钙素相当 .The methionine at position 38 in the Trx sCT Gly was mutated into alanine and the mutated gene was cloned into the expressing vector pET30a.It was expressed in high level in E.coli BL21(DE3).The fusion protein(Trx sCT Gly) was purified with ThioBind affinity resin and it was found that the mutation of methionine to alanine does not affect the recombinant protein to the ThioBind resin.The fusion protein was cleaved by cyanogen bromide in presence of 3 mol/L urea in 70% formic acid for 48 hours in room temperature,at least 80% of the recombinant protein was cleaved.The cleaved peptide sCT Gly was purified by CM cellulose absorption and the peptide had the purity about 92%.N terminal amino acid sequencing result of sCT Gly shows that at least 10 amino acid residues are the same as natural sCT.No deamidation reaction occurred in the preparation .The p I measured by capillary electrophoresis was 6.46.Modi Tof mass spectra result showed that the molecular weight was 3 492.Bioassay in rats showed that special bioactivity was 190 IU/mg,almost the same as that of natural human calcitonin.

关 键 词：重组降钙素前体 制备 性质 基因工程药物 

分 类 号：TQ464.7[化学工程—制药化工] Q78[生物学—分子生物学]