人甲状旁腺素(1-34)衍生物在大肠杆菌中表达、纯化及其生物学活性

为研究Gly hPTH(1 34)衍生物的生物学活性 ,用重叠PCR方法合成编码hPTH(1 34)的DNA片段 ,克隆到融合表达载体pGEX 2T的缩短型谷胱甘肽转移酶基因GST6 9△的 3′末端 ,构建正确读码框架的融合基因 .在两个基因间引入蛋白质羟胺切割位点序列 ,转入E .coliJM10 9中 ,IPTG诱导表达 .该融合蛋白的表达量占菌体总蛋白的 2 0 %以上 ,主要以包涵体形式存在 ,盐酸羟胺切割表达产物 .分析表明 ,80 %左右的融合蛋白被裂解为GST6 9△和Gly hPTH(1 34) .经分子筛柱层析和反相层析分离纯化获得重组Gly hPTH(1 34)衍生物 ,纯度达 98%以上 ,回收率约为 10mg/升发酵液 ,分子量为 4 177,等电点 (pI)为 8 4 0 ,N端 16个氨基酸 ,除第一个为甘氨酸外 ,其余与天然hPTH(1 34)序列一致 .Western印迹结果表明 ,Gly hPTH(1 34)衍生物具有hPTH(1 34)的免疫学活性 .体外活性测定结果表明 ,Gly hPTH(1 34)衍生物能刺激人成骨细胞HOSTE85增殖、增加细胞内胶原合成、ALP活性增高和cAMP生成量增加 ,并呈量效关系 ,提示它具有与化学合成的hPTH(1 34)相同的生物学活性 ,N端多一个Gly对其活性无明显影响 .

Expression,Purification and Biological Activity of a Recombinant Human Parathyroid Hormone(1-34) Analogue in E. coli

Recombinant human parathyroid hormone(1-34) analogue with a amino-terminal extension of glycine,Gly-hPTH(1-34),was obtained from E. coli using a hydroxylamine cleavage fusion protein strategy.The DNA fragment encoding Asn-Gly-hPTH(1-34) was synthesized by overlapping PCR and cloned into the 3′ end of the truncated GST gene(GST69△) in expression vector pGEX-2T to construct the GST69△ and Asn-Gly-hPTH(1-34) fusion expression vector in which there was a hydroxylamine site between the two genes.The fusion protein was expressed in the inclusion bodies in E.coli JM109,and yield rate was about 20% of the total bacteria proteins.The inclusion bodies were dissolved with 8.0 mol/L urea-glycine buffer and subjected to site-specific cleavage with hydroxylamine.SDS-PAGE analysis showed that about 80% the fusion protein was cleaved to GST69△ and Gly-hPTH(1-34) analogue.Over 98% purity of recombinant Gly-hPTH(1-34) was obtained through Sephadex G-50 and RP-HPLC purification procedures.N-terminal amino acids were determined by sequencing,isoelectric point(pI) determined by capillary electrophoresis and molecular weight measured by Maldi-Tof mass spectra.The results showed that 16 amino acid residues were the same as natural hPTH(1-34) except the first one was glycine,pI 8.40,molecular weight 4177.The bioactivity indicated that Gly-hPTH(1-34) analogue could stimulate remarkable proliferation of human osteoblast cells(HOSTE85),increase intracellular collagen sythesis, enhance intracellular cAMP production and celluar alkaline phosphatase(ALP) activity.Recombinant Gly-PTH(1-34) analogue has almost same bioactivity and immunoactivity of the chemically sythetic hPTH(1-34) and an amino-terminal extension of glycine has no effect on bioactivity of PTH(1-34).