Xiu Zhao-Yang, Zhou He-Yue, Yu Ying, Dai Jin-Feng, Chen Chang-Qing
Research Center of Biotechnology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Science, Shanghai 200233, China.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2002 Jul;34(4):469-74.
An engineering E.coli strain, BL21 (DE3)/pGEX-4T hPTH (1-34), was constructed by oligonucleotide annealing and PCR amplifying the target gene, then ligating it with pGEX-4T-3 vector and transferring into BL21 host. The yield of soluble fusion protein of GST-hPTH(1-34) expressed from BL21(DE3)/pGEX-4T hPTH(1-34) is about 10 g/L after high-density, high expression culture and purification by affinity chromatography. Following the simple digestion of enterokinase, about 0.6 g/L intact hPTH (1-34) was harvested. The product is checked by HPLC MS and N-terminus sequence analysis. The purified recombinant hPTH(1-34) stimulated adenylate cyclase in rabbit renal cortical cell membranes to exactly the same extent as synthetic human parathyroid hormone standards, indicating that the recombinant product has full biological activity.
通过寡核苷酸退火和PCR扩增目标基因，构建了工程大肠杆菌菌株BL21(DE3)/pGEX-4T hPTH(1-34)，然后将其与pGEX-4T-3载体连接并转入BL21宿主。经高密度、高表达培养及亲和层析纯化后，BL21(DE3)/pGEX-4T hPTH(1-34)表达的可溶性融合蛋白GST-hPTH(1-34)产量约为10 g/L。经肠激酶简单消化后，收获了约0.6 g/L完整的hPTH(1-34)。通过HPLC MS和N端序列分析对产物进行检测。纯化的重组hPTH(1-34)刺激兔肾皮质细胞膜中的腺苷酸环化酶，其程度与合成人甲状旁腺激素标准品完全相同，表明重组产物具有完全的生物活性。
