High yield and secretion of recombinant human apolipoprotein AI in Pichia pastoris》.pdfVIP

Protein Expression and PuriWcation 46 (2006) 337–342 /locate/yprep High yield and secretion of recombinant human apolipoprotein AI in Pichia pastoris Mei-qing Feng 1, Qin-sheng Cai 1, Da-xin Song, Ji-bin Dong, Pei Zhou ¤ School of Pharmacy, Fudan University, Yi Xue Yuan Road 138#, Shanghai 200032, China Received 5 July 2005, and in revised form 3 November 2005 Available online 12 December 2005 Abstract Apolipoprotein AI (ApoAI) is an important apolipoprotein in plasma and is known to have various physiological functions suitable for pharmaceutical applications. Human blood has been the only source of this protein for research and large-scale applications. To obtain large amounts of ApoAI a Pichia pastoris expression system was Wrst used to obtain a high level of expression of secreted, recom- binant protein. The human gene encoding ApoAI was inserted into the secretion vector pPIC9K and used to transform P. pastoris GS115. AP16, a high expression transformant with high G418 resistance, was obtained. After induction with methanol, the expression level of rhApoAI (recombinant human ApoAI) was 160 mg/L in a 14 L fermentor. RhApoAI was puriWed by cold acetone precipitation followed by Q-Sepharose fast Xow ion exchange column chromatography with 60% recovery. The N-terminal amino acid sequence and molecular weight (mass spec.) of rhApoAI are identical to native human ApoAI. PuriWed rhApoAI has speciWc binding activity with liver cells SMC7721 and binding can be inhibited by native human ApoAI. © 2005 Elsevier Inc. All rights reserved. Keywords: Rec