Production of recombinant human midkine in yeast, Pichia pastoris

Recombinant human midkine was expressed in the cells of Pichia pastoris und er the control of the AOX1 gene promoter. The expression of midkine was eff iciently induced by methanol in a high cell density fermentation. Approxima tely 0.3 g/l culture of midkine accumulated in the cells by 72 h after indu ction. 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 8M guanidine hydroch loride, 10 mM dithiothreitol, I mM EDTA and 50 mM Tris-Cl, and then, midkin e was renatured by dialysis at high concentration against the buffer soluti on (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-terminu s and the amino acid composition of midkine were the same as those of Met-m idkine that has a methionine residue at the amino-teminus. Mass spectrometr y 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 Chi nese hamster ovary (CHO) cells.