High yield and secretion of recombinant human apolipoprotein AI in Pichia pastoris.
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 first used to obtain a high level of expression of secreted, recombinant 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 14L fermentor. RhApoAI was purified by cold acetone precipitation followed by Q-Sepharose Fast Flow 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. Purified rhApoAI has specific binding activity with liver cells SMC7721 and binding can be inhibited by native human ApoAI.
Feng
Mei-qing
MQ
School of Pharmacy, Fudan University, Yi Xue Yuan Road 138#, Shanghai 200032, China. fmeiqing@126.com
Cai
Qin-sheng
QS
Song
Da-xin
DX
Dong
Ji-bin
JB
Zhou
Pei
P
D016632:Apolipoprotein A-I / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D010843:Pichia; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D016632:Apolipoprotein A-I; D011994:Recombinant Proteins