[Expression of human apolipoprotein A- I in baculovirus-insect cell system].
Apolipoprotein A- I is the major apolipoprotein in high-density lipoprotein known to have a wide range of physiological functions, the best-studied one of which is in regulating cholesterol metabolism and preventing arteriosclerosis. Human blood has been the only source of this protein. To facilitate further research and application, it is essential to produce it through genetic engineering. In the current research, the baculovirus-insect cell system was used to overexpress human apolipoprotein A- I . Two recombinant baculoviruses were constructed. The first one expressed a pro form of apoA- I lacking native signal peptide. The recombinant protein was found to remain mainly inside cells in the early phase of infection, while being largely excreted to the medium late in infection. The second one used a heterologous signal peptide, snake phospholipase A2 inhibitor alpha subunit signal peptide, to lead the secretion of mature apoA- I. In contrast to the first virus, recombinant apoA- I was found in the culture medium at the early phase of virus infection. The mature apoA- I was purified from culture medium using Phenyl Sepharose hydrophobic interaction chromatography (HIC) and eluted with water and Propylene. This work shows that snake phospholipase A2 inhibitor a subunit signal peptide can be used to secret human apoA- I in insect cells, but the efficiency of its secretion is limited when the expression level is high.
Zhu
Yuan-E
YE
Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
Xu
Hui-Bin
HB
Zhao
Zhi-An
ZA
Zhong
Jiang
J
D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics*; D001798:Blood Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002845:Chromatography; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012911:Snakes; D018411:Spodoptera / Q000166:cytology*
C505693:APOA1 protein, human; D016632:Apolipoprotein A-I; D001798:Blood Proteins; C400481:PLIalpha; D011994:Recombinant Proteins