Abstract
Recombinant human midkine (rh-midkine) was efficiently produced in Pichia pastoris using the pre–pro secretion signal of yeast α-mating factor under the control of the AOX1 promoter. The pep4 host SMD1168 was used. The expression was induced at pH 3 and 20 °C in high cell-density fermentation and approximately 360 mg rh-midkine was secreted into 1 L of medium. The authentic midkine could be obtained after one-step purification. Mass spectrometry of purified rh-midkine demonstrated a single large signal for the molecular ion [M+H] + at 13241.2 m/z. This mass is identical to the authentic, unmodified human midkine. The precursor of rh-midkine was correctly processed in P. pastoris cells, yielding mature rh-midkine. Mass spectrometry detected no yeast-specific O-mannosylations in the purified midkine preparations. The circular dichroic spectrum indicated only a negative Cotton effect at 215 nm. Only β-structures were indicated for the rh-midkine molecule in solution. Purified rh-midkine was active in a cell-proliferation assay.
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