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    • 4. 发明申请
    • PROCESS FOR BACTERIAL PRODUCTION OF POLYPEPTIDES
    • 多肽生产细菌的方法
    • WO1998018946A1
    • 1998-05-07
    • PCT/US1997018383
    • 1997-10-09
    • GENENTECH, INC.
    • GENENTECH, INC.JOLY, John, C.SWARTZ, James, R.
    • C12N15/70
    • C12N9/0051C07K1/1133C07K14/245C07K14/65C07K2319/00C12N9/90C12N15/70C12P21/02
    • A process is provided for producing a heterologous polypeptide in bacteria. This process comprises, in a first step, culturing bacterial cells that lack their native pstS gene and comprise nucleic acid encoding a PstS variant having an amino acid variation within the phosphate-binding region of the corresponding native PstS, nucleic acid encoding a DsbA or DsbC protein, nucleic acid encoding the heterologous polypeptide, a signal sequence for secretion of both the DsbA or DsbC protein and the heterologous polypeptide, an inducible promoter for the nucleic acid encoding the DsbA or DsbC protein, and an alkaline phosphatase promoter for the nucleic acid encoding the heterologous polypeptide. The nucleic acid encoding a PstS variant is under the transcriptional control of the wild-type pstS gene promoter. The culturing step is conducted under conditions whereby expression of the nucleic acid encoding the DsbA or DsbC protein is induced prior to induction of the expression of the nucleic acid encoding the heterologous polypeptide, whereby the culturing takes place in a culture medium at a concentration of inorganic phosphate in the medium that during all phases of cell growth is above the level at which the cells are starved for phosphate, and whereby either both the heterologous polypeptide and the DsbA or DsbC protein are secreted into the periplasm of the bacteria or the heterologous polypeptide is secreted into the medium in which the bacterial cells are cultured. The second step of the process involves recovering the heterologous polypeptide from the periplasm or the culture medium.
    • 提供了用于在细菌中产生异源多肽的方法。 该方法在第一步中包括培养缺乏其天然pstS基因的细菌细胞,并且包含编码在相应天然PstS的磷酸结合区域内具有氨基酸变异的PstS变体的核酸,编码DsbA或DsbC的核酸 蛋白质,编码异源多肽的核酸,用于分泌DsbA或DsbC蛋白质和异源多肽的信号序列,用于编码DsbA或DsbC蛋白质的核酸的诱导型启动子,以及用于核酸编码的碱性磷酸酶启动子 异源多肽。 编码PstS变体的核酸在野生型pstS基因启动子的转录控制下。 培养步骤是在诱导编码异源多肽的核酸表达之前诱导编码DsbA或DsbC蛋白的核酸的表达的条件下进行的,由此培养在培养基中以无机的浓度进行 在培养基中,在细胞生长的所有阶段都高于细胞饥饿磷酸盐的水平,并且由此异源多肽和DsbA或DsbC蛋白质分泌到细菌的周质或异源多肽中的是 分泌到培养细菌细胞的培养基中。 该方法的第二步包括从周质或培养基中回收异源多肽。