Eukaryotic Expression and Purification of Native Form of Mouse Midkine from Pichia pastoris.
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.
School of Pharmacy, Shanghai Jiao Tong University, Room 6214, 800 Dongchuan Road, Minhang District, Shanghai, 200240, People's Republic of China.
Jin
J
Gao
School of Agriculture and Biology, Shanghai Jiao Tong University, Room 3409, 800 Dongchuan Road, Minhang District, Shanghai, 200240, People's Republic of China.
Haixia
H
Wang
School of Pharmacy, Shanghai Jiao Tong University, Room 6214, 800 Dongchuan Road, Minhang District, Shanghai, 200240, People's Republic of China.
Jingjing
J
Li
General regeneratives Limited, Shanghai, 200203, People's Republic of China.
Shixiang
S
Jia
School of Pharmacy, Shanghai Jiao Tong University, Room 6214, 800 Dongchuan Road, Minhang District, Shanghai, 200240, People's Republic of China. weisjtulab@126.com.
Wei
W
Han
School of Agriculture and Biology, Shanghai Jiao Tong University, Room 3409, 800 Dongchuan Road, Minhang District, Shanghai, 200240, People's Republic of China. yansjtulab@gmail.com.
Yan
Y
Yu
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D016207:Cytokines / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006801:Humans; D051379:Mice; D000078792:Midkine; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D016207:Cytokines; D011994:Recombinant Proteins; D000078792:Midkine
Fermentation; HPLC; Ion exchange chromatography; Midkine; Pichia pastoris