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Cited by 3Biochemical Acid Research StudiesViral Infectious Diseases and Gene Expression in InsectsEnzyme Structure and Function

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Component co-expression and purification of recombinant human pyruvate dehydrogenase complex from baculovirus infected SF9 cells

Jiang, Yong, Wang, Juan, Zhang, Guofeng, Oza, Khyati, Myers, Linda, Holbert, Marc A., Sweitzer, Sharon·2014·Protein Expression and Purification
DOI 10.1016/j.pep.2014.02.002
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Abstract

The mammalian pyruvate dehydrogenase complex (PDC) is a multi-component mitochondrial enzyme that plays a key role in the conversion of pyruvate to acetyl-CoA connecting glycolysis to the citric acid cycle. Recent studies indicate that targeting the regulation of PDC enzymatic activity might offer therapeutic opportunities by inhibiting cancer cell metabolism. To facilitate drug discovery in this area, a well defined PDC sample is needed. Here, we report a new method of producing functional, recombinant, high quality human PDC complex. All five components were co-expressed in the cytoplasm of baculovirus-infected SF9 cells by deletion of the mitochondrial localization signal sequences of all the components and E1a was FLAG-tagged to facilitate purification. The protein FLAG tagged E1a complex was purified using FLAG-M2 affinity resin, followed by Superdex 200 sizing chromatography. The E2 and E3BP components were then Lipoylated using an enzyme based in vitro process. The resulting PDC is over 90% pure and homogenous. This non-phosphorylated, lipoylated human PDC was demonstrated to produce a robust detection window when used to develop an enzyme coupled assay of PDHK.

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Metadata

Authors
Jiang, Y., Wang, J., Zhang, G., Oza, K., Myers, L., Holbert, M. A., & Sweitzer, S.
Affiliations
GlaxoSmithKline (United States) (US)
Corresponding author
Jiang, Yong
ORCID
Venue
Protein Expression and Purification
Publisher
Elsevier BV
Published
2014-05
Volume
97
Pages
9-16
ISSN
1046-5928 (ISSN-L)
DOI
10.1016/j.pep.2014.02.002
OpenAlex
W2137306790
Cited by
3
References
0
Citations by year
2025: 1 · 2023: 1 · 2020: 1
FWCI
0.00
Type
journal article
Language
en
Open access
closed
Locations
  • Protein Expression and Purification (publishedVersion)
  • PubMed (publishedVersion)
Funders
GlaxoSmithKline
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Biochemical Acid Research Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords
pyruvate dehydrogenase complex, recombinant protein expression, baculovirus-infected insect cells, Sf9 cells, lipoylation, cancer metabolism, drug discovery
MeSH terms
Pyruvate Dehydrogenase Acetyl-Transferring Kinase, Animals, Cloning, Molecular, Humans, Phosphorylation, Pyruvate Dehydrogenase Complex, Recombinant Proteins, Gene Expression, Baculoviridae, Protein Serine-Threonine Kinases, Sf9 Cells

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