Suzuki Y, Yabuta M, Ohsuye K
Suntory Institute for Medicinal Research and Development, Gunma, Japan.
Appl Environ Microbiol. 1998 Feb;64(2):526-9. doi: 10.1128/AEM.64.2.526-529.1998.
Expression of the synthetic human parathyroid hormone 1-34 [hPTH(1-34)] gene by a gene fusion strategy was demonstrated. hPTH(1-34) was produced at the C terminus of the partner peptides involving amino acids 1 to 97, 1 to 117, or 1 to 139 of a modified Escherichia coli beta-galactosidase by linker peptides containing oligohistidine of different lengths. The fusion proteins in the inclusion bodies were rendered soluble with urea and subjected to site-specific cleavage with the secretory type yeast Kex2 protease. Optimal expression and enzymatic processing were achieved in the fusion protein beta G-117S4HPT, constructed from amino acids 1 to 117 of beta-galactosidase and the linker of HHHHPGGSVKKR. The fusion protein accumulated more than 20% of the E. coli total protein. The hPTH(1-34) was purified up to 99.5% with a good yield of 0.5 g/liter of culture. The purified product was identified as intact hPTH(1-34) by amino acid analysis and N-terminal sequencing.
通过基因融合策略证明了合成人甲状旁腺激素1 - 34 [hPTH(1 - 34)]基因的表达。hPTH(1 - 34)在伴侣肽的C末端产生，这些伴侣肽包含经修饰的大肠杆菌β-半乳糖苷酶的1至97、1至117或1至139个氨基酸，并通过含有不同长度寡组氨酸的接头肽连接。包涵体中的融合蛋白用尿素处理使其溶解，并用分泌型酵母Kex2蛋白酶进行位点特异性切割。在由β-半乳糖苷酶的1至117个氨基酸和HHHHPGGSVKKR接头构建的融合蛋白βG - 117S4HPT中实现了最佳表达和酶促加工。该融合蛋白积累量超过大肠杆菌总蛋白的20%。hPTH(1 - 34)以0.5 g/升培养物的良好产量纯化至99.5%。通过氨基酸分析和N端测序鉴定纯化产物为完整的hPTH(1 - 34)。
