Wang Sue-Hong, Yang Tien-Shuh, Lin Shiang-Ming, Tsai Ming-Shiun, Wu Shinn-Chih, Mao Simon J T
Jen-Te Junior College of Medicine Nursing and Management, Miaoli, Taiwan.
Protein Expr Purif. 2002 Jun;25(1):41-9. doi: 10.1006/prep.2001.1607.
Recombinant porcine lactoferrin (rPLF) was synthesized in Pichia pastoris using a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene. Strains expressing rPLF with its own signal sequence or with that from the yeast alpha-mating factor (alpha-MF) were able to produce and secrete rPLF, but levels were consistently higher using alpha-MF constructs. In contrast, P. pastoris strains that expressed rPLF without a signal sequence produced the protein in an insoluble intracellular form. Increasing the initial pH of shake-flask culture medium from 6.0 to 7.0 or adding ferric ions to the medium (to 100 microM) resulted in significant improvements in expression of rPLF from P. pastoris. Expression levels (approximately 12 mg/L) were much higher than those observed from Saccharomyces cerevisiae strains (1-2 mg/L). P. pastoris-secreted rPLF was isolated and purified via a one-step simple procedure using a heparin column. The molecular size (78 kDa), isoelectric point (8.8-9.0), N-terminal amino acid sequence, and iron-binding capability of rPLF were each similar to that of native milk PLF.
利用甘油醛-3-磷酸脱氢酶基因的组成型启动子，在毕赤酵母中合成重组猪乳铁蛋白（rPLF）。表达带有自身信号序列或酵母α-交配因子（α-MF）信号序列的rPLF的菌株能够产生并分泌rPLF，但使用α-MF构建体时水平始终更高。相比之下，不带有信号序列表达rPLF的毕赤酵母菌株以不溶性细胞内形式产生该蛋白。将摇瓶培养基的初始pH从6.0提高到7.0或向培养基中添加铁离子（至100μM），可显著提高毕赤酵母中rPLF的表达。表达水平（约12mg/L）远高于酿酒酵母菌株（1-2mg/L）中观察到的水平。通过使用肝素柱的一步简单程序分离并纯化毕赤酵母分泌的rPLF。rPLF的分子大小（78kDa）、等电点（8.8-9.0）、N端氨基酸序列和铁结合能力均与天然乳PLF相似。
