High yield and purification of recombinant human apolipoprotein E3 in Pichia pastoris.
Apolipoprotein E3 (ApoE3) is an important apolipoprotein in plasma and plays a critical role in lipid transport and cholesterol homeostasis. As the only natural source of this protein, human blood cannot provide large-scale ApoE3 for research and applications. Therefore, in our study, a Pichia pastoris expression system was first used to obtain a high-level expression of secreted, recombinant human ApoE3 (rhApoE3). The full-length sequence encoding ApoE3, gained by RT-PCR, was inserted into the pPICZalphaC vector and transformed into P. pastoris strain X33, and then the high expression transformants with zeocin resistance were obtained. The growth conditions of the transformant strains were optimized in 50ml conical tubes including pH and inducing time. After induction with methanol, the expression level of rhApoE3 was 120 mg/L in 80 L fermentor. RhApoE3 was purified more than 94% purity using SP Sepharose ion exchange chromatography and source 30RPC. A preliminary biochemical characterization of purified rhApoE3 was performed by analyzing the ability of inhibiting PDGF-induced proliferation of rat coronary artery smooth muscle cells (SMCs), and the results demonstrated that the function of purified rhApoE3 was similar to natural human ApoE3.
Su
Manman
M
Department of Biological Engineering, College of Pharmacy, Jilin University, 1266 Fu Jin Road, Changchun 130021, China.
Xu
Tianmin
T
Wang
Doudou
D
Zhou
Yulai
Y
Niu
Chao
C
Yan
Weiqun
W
D000818:Animals; D053318:Apolipoprotein E3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D032389:Myocytes, Smooth Muscle; D010843:Pichia / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013997:Time Factors
D053318:Apolipoprotein E3; D011994:Recombinant Proteins