Publication: Expression of human pyruvate carboxylase in insect cells using the baculovirus system
Issued Date
2004-03-26
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ISSN
0006291X
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2-s2.0-1542345765
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Mahidol University
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SCOPUS
Bibliographic Citation
Biochemical and Biophysical Research Communications. Vol.316, No.1 (2004), 177-181
Suggested Citation
Natthida Petchampai, Vichai Boonsaeng, John C. Wallace, Sarawut Jitrapakdee Expression of human pyruvate carboxylase in insect cells using the baculovirus system. Biochemical and Biophysical Research Communications. Vol.316, No.1 (2004), 177-181. doi:10.1016/j.bbrc.2004.02.028 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/21212
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Title
Expression of human pyruvate carboxylase in insect cells using the baculovirus system
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Abstract
Constructs containing cDNA encoding human pyruvate carboxylase (PC) with and without a hexahistidine (6x His) tag at the N-terminal of the mature enzyme have been cloned under the control of the polyhedrin promoter. These two constructs were co-transfected with the baculovirus genome into Sf9 cells to produce recombinant baculoviruses harbouring human PC cDNA. The expression of human PC under the control of the polyhedrin promoter was found to be at its highest level at 4 days post-infection. The expressed material accounted for up to 70% of total cellular protein with 5% of this expressed material being found in the soluble fraction. The recombinant human 6x His-PC isolated with a purity of ∼50% using a Ni-NTA agarose column was found to have the specific activity of 7U/mg, which was similar to that produced from a 293T stable line [Biochem. Biophys. Res. Commun. 266 (1999) 512]. This is the first report of a heterologous expression system for recombinant human PC. © 2004 Elsevier Inc. All rights reserved.