Abstract:
:Acyl coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his-tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally occurring fluorescent cis-parinaroyl-CoA with very high affinity (K(d)=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his-tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor-4alpha (HNF-4alpha), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4alpha were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4alpha (intermolecular distance of 73 A) at high affinity (K(d)=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Petrescu AD,Huang H,Hostetler HA,Schroeder F,Kier ABdoi
10.1016/j.pep.2007.11.010subject
Has Abstractpub_date
2008-04-01 00:00:00pages
184-93issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(07)00294-Xjournal_volume
58pub_type
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journal_title:Protein expression and purification
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