Abstract
To confirm the treating effectiveness of midkine as an articular protective agent, mouse midkine (mMK) was produced for the pre-clinic long-term studies in mice. The protein was expressed under the control of the AOX1 gene promoter in Pichia pastoris, X-33 strain, and secreted into fermentation broth through high-density fermentation. Approximately 380 mg mMK, containing authentic and truncated forms, was secreted into 1 liter induction medium and 280 mg mMK was obtained after one-step purification on a 50 ml SP Sepharose Fast Flow column. The purified protein was characterized and identified to be the mature, authentic form of mMK. N-terminal five amino acid sequence was determined to be K-K-K-E-K. SDS-PAGE analysis indicated that the molecular weight of the product was about 13 KDa. The purity of the purified rmMK protein was determined to be 99% by high performance liquid chromatography. The biological activity of final product was verified via migration assay on osteoblast-like UMR-106 cells.
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