Production of Recombinant Human Midkine in Yeast, Pichia pastoris

概要

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Recombinant human midkine was expressed in the cells of Pichia pastoris under the control of 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.
社団法人日本生物工学会の論文
2000-10-25