A process for the production of a physiologically active peptide (a target peptide) containing cysteine residue, comprising the steps of:
(1) culturing Escherichia coli transformed with a plasmid capable of expressing a fusion protein under control of a promoter of E. coli origin or a promoter of a phage origin, wherein the fusion protein is represented by the following formula:
A - L - B
wherein B represents a target peptide containing cysteine residue; A represents a partner polypeptide consisting of 90 to 220 amino acid residue but not containing cysteine residue; and L represents a linker amino acid residue positioned between C-terminal of the partner polypeptide and N-terminal of the target peptide wherein the same amino acid as the linker amino acid is not present in the target peptide, and the linker amino acid is selected so that the peptide bond between the C-terminal of the linker amino acid and the N-terminal of the target peptide is claimed by a protease or the linker amino acid is selectively degraded by a chemical substance;
(2) disrupting the cultured cells and obtaining an insoluble fraction containing the fusion protein;
(3) solubilizing the fusion protein with a solubilizing agent, and treating the solubilized fusion protein with the protease or the chemical substance to liberate the target peptide, and isolating the target peptide.
一种生产含有半胱
氨酸残基的生理活性肽(目标肽)的工艺,包括以下步骤
(1) 培养用质粒转化的大肠杆菌,该质粒能在大肠杆菌源性启动子或噬菌体源性启动子的控制下表达融合蛋白,其中融合蛋白由下式表示:
A - L - B
其中 B 代表含有半胱
氨酸残基的目标肽;A 代表由 90 至 220 个
氨基酸残基组成但不含半胱
氨酸残基的伙伴
多肽;L 代表位于伙伴
多肽的 C 末端和目标肽的 N 末端之间的连接
氨基酸残基,其中与连接
氨基酸相同的
氨基酸不存在于目标肽中,连接
氨基酸的选择是为了使连接
氨基酸的 C 末端和目标肽的 N 末端之间的肽键被
蛋白酶破坏或连接
氨基酸被
化学物质选择性降解;
(2) 破坏培养细胞并获得含有融合蛋白的不溶部分;
(3) 用
增溶剂增溶融合蛋白,用
蛋白酶或
化学物质处理增溶的融合蛋白以释放目的肽,并分离目的肽。