Production of native recombinant human midkine in the yeast, Pichia pastoris.
Recombinant human midkine (rh-midkine) was efficiently produced in Pichia pastoris using the pre-pro secretion signal of yeast alpha-mating factor under the control of the AOX1 promoter. The pep4 host SMD1168 was used. The expression was induced at pH 3 and 20 degrees C in high cell-density fermentation and approximately 360 mg rh-midkine was secreted into 1L 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 beta-structures were indicated for the rh-midkine molecule in solution. Purified rh-midkine was active in a cell-proliferation assay.
Murasugi
Akira
A
Meiji Cell Technology Center, Meiji Milk Products Co, Ltd, 540 Naruda, Odawara, Kanagawa 250-0862, Japan. AKIRA_MURASUGI@MEIJI-MILK.COM
Tohma-Aiba
Yumiko
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D002455:Cell Division; D002942:Circular Dichroism; D016207:Cytokines; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008358:Mannose / Q000737:chemistry; D013058:Mass Spectrometry; D000078792:Midkine; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D002352:Carrier Proteins; D016207:Cytokines; D011994:Recombinant Proteins; D000078792:Midkine; D004247:DNA; D008358:Mannose