RESUMO
The secretion signals of Saccharomyces cerevisiae alpha mating factor, human midkine itself, and Pichia pastoris acid phosphatase, were tried for the expression of human midkine under the control of the AOX1 gene promoter in P. pastoris. Approximately 28 mg/l, 1.5 mg/l, and 0.2 mg/l of midkine were secreted by using the a mating factor pre-pro-sequence, the midkine signal sequence, and the phosphatase signal sequence in flask cultures, respectively.
Assuntos
Proteínas de Transporte/biossíntese , Citocinas , Fatores de Crescimento Neural/biossíntese , Pichia/metabolismo , Sinais Direcionadores de Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Regulação Fúngica da Expressão Gênica , Vetores Genéticos/genética , Humanos , Midkina , Proteínas Mitocondriais , Dados de Sequência Molecular , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Oxirredutases/genética , Pichia/química , Pichia/genética , Proteínas de Plantas , Sinais Direcionadores de Proteínas/fisiologia , Transformação Genética/fisiologiaRESUMO
Recombinant human midkine was expressed in the cells of Pichia pastoris under the control of the AOX1 gene promoter. The expression of midkine was efficiently induced by methanol in a high cell density fermentation. Approximately 0.3 g/l culture of midkine accumulated in the cells by 72 h after induction. When the cells were disrupted, midkine was recovered in an insoluble form, and was insoluble even in the presence of 7 M urea. The precipitate was dissolved in the buffer solution (pH 8) containing 8 M guanidine hydrochloride, 10 mM dithiothreitol, 1 mM EDTA and 50 mM Tris-Cl, and then, midkine was renatured by dialysis at high concentration against the buffer solution (pH 8) containing 0.5 M sodium chloride and 20 mM Tris-Cl. The renatured midkine was recovered using a SP-Sepharose column, and purified further by Heparin-Sepharose column chromatography. Approximately 64 mg/l culture of the purified midkine was obtained. The amino acid sequence of amino-terminus and the amino acid composition of midkine were the same as those of Met-midkine that has a methionine residue at the amino-teminus. Mass spectrometry of purified Met-midkine showed a mass of 13370.7 Da (average), almost the theoretical mass for it. The Met-midkine enhanced the proliferation of Chinese hamster ovary (CHO) cells.
