Petrescu Anca D, Huang Huan, Hostetler Heather A, Schroeder Friedhelm, Kier Ann B
Department of Physiology and Pharmacology, Texas A&M University, 4467 TAMU, TVMC, College Station, TX 77843-4467, USA.
Protein Expr Purif. 2008 Apr;58(2):184-93. doi: 10.1016/j.pep.2007.11.010. Epub 2007 Dec 4.
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.
酰基辅酶A结合蛋白（ACBP）被认为可在细胞内转运脂肪酰基辅酶A，促进其代谢。在本研究中，通过在C末端插入组氨酸（his）标签，制备了一种新的小鼠重组ACBP，以便通过镍亲和层析进行高效纯化。将his标签插入C末端是因为ACBP是一种小分子（10 kDa）蛋白，其结构和活性对N末端的氨基酸取代敏感。his标签对ACBP的结构或配体结合亲和力及特异性没有或几乎没有影响。His-ACBP以非常高的亲和力（K(d)=2.15 nM）结合天然存在的荧光顺式十八碳四烯酰辅酶A，但对非酯化的顺式十八碳四烯酸没有亲和力。为了确定C末端his标签的存在是否改变了ACBP与其他蛋白质的相互作用，研究了其与肝细胞核因子-4α（HNF-4α）的直接结合，HNF-4α是一种调节参与脂质代谢基因转录的核受体。His-ACBP和HNF-4α分别用Cy5和Cy3标记，并通过一种新型的荧光共振能量转移（FRET）结合试验确定直接相互作用。FRET分析表明，His-ACBP以与天然ACBP相似的高亲和力（K(d)=64-111 nM）直接与HNF-4α相互作用（分子间距离为73 Å）。his标签对ACBP与利用或形成脂肪酰辅酶A的微粒体酶相互作用并刺激其活性的能力也没有影响。因此，C末端带有his标签的ACBP保持了与未标记天然蛋白非常相似的结构和功能特征，可用于需要纯重组ACBP的进一步体外实验。
