title: High-yield soluble expression, purification and characterization of human steroidogenic acute regulatory protein (StAR) fused to a cleavable Maltose-Binding Protein (MBP).
abstract: Steroidogenic acute regulatory protein (StAR) is responsible for the rapid delivery of cholesterol to mitochondria where the lipid serves as a source for steroid hormones biosynthesis in adrenals and gonads. Despite many successful investigations, current understanding of the mechanism of StAR action is far from being completely clear. StAR was mostly obtained using denaturation/renaturation or in minor quantities in a soluble form at decreased temperatures that, presumably, limited the possibilities for its consequent detailed exploration. In our hands, existing StAR expression constructs could be bacterially expressed almost exclusively as insoluble forms, even upon decreased expression temperatures and in specific strains of Escherichia coli, and isolated protein tended to aggregate and was difficult to handle. To maximize the yield of soluble protein, optimized StAR sequence encompassing functional domain STARD1 (residues 66-285) was fused to the C-terminus of His-tagged Maltose-Binding Protein (MBP) with the possibility to cleave off the whole tag by 3C protease. The developed protocol of expression and purification comprising of a combination of subtractive immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography allowed us to obtain up to 25 mg/1 L culture of completely soluble StAR protein, which was (i) homogenous according to SDS-PAGE, (ii) gave a single symmetrical peak on a gel-filtration, (iii) showed the characteristic CD spectrum and (iv) pH-dependent ability to bind a fluorescently-labeled cholesterol analogue. We conclude that our strategy provides fully soluble and native StAR protein which in future could be efficiently used for biotechnology and drug discovery aimed at modulation of steroids production.
authors.affiliation: A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia. Electronic address: nikolai.sluchanko@mail.ru.
authors.forename: Nikolai N
authors.initials: NN
authors.lastname: Sluchanko
authors.affiliation: A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia; Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russia.
authors.forename: Kristina V
authors.initials: KV
authors.lastname: Tugaeva
authors.affiliation: Research Institute for Physical Chemical Problems, Belarusian State University, Minsk, Belarus.
authors.forename: Yaroslav V
authors.initials: YV
authors.lastname: Faletrov
authors.affiliation: A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia; A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia.
authors.forename: Dmitrii I
authors.initials: DI
authors.lastname: Levitsky
mesh_terms: D000595:Amino Acid Sequence; D002784:Cholesterol / Q000737:chemistry; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D058619:Maltose-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011485:Protein Binding; D012995:Solubility; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D058619:Maltose-Binding Proteins; D010750:Phosphoproteins; D000098682:Steroidogenic Acute Regulatory Protein; D002784:Cholesterol
keywords: Cholesterol; Fluorescence; Maltose-Binding Protein; Soluble expression; Steroid hormones; Steroidogenic acute regulatory protein