title: Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones.
abstract: Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred to as monomeric in this work) in a straightforward and facile manner will obviate protein--production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove.
authors.affiliation: Institute for Research and Technology-Thessaly (I.RE.TE.TH.) Centre for Research & Technology Hellas (CE.R.TH.), 95 Dimitriados & Pavlou Mela Street, GR 38333, Volos, Greece; Laboratory of Biochemistry, Faculty of Veterinary Science, University of Thessaly, GR-43100 Karditsa, Greece.
authors.forename: Asterios I
authors.initials: AI
authors.lastname: Grigoroudis
authors.affiliation: Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208, United States.
authors.forename: Campbell
authors.initials: C
authors.lastname: McInnes
authors.affiliation: Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208, United States.
authors.forename: Padmavathy Nandha
authors.initials: PN
authors.lastname: Premnath
authors.affiliation: Institute for Research and Technology-Thessaly (I.RE.TE.TH.) Centre for Research & Technology Hellas (CE.R.TH.), 95 Dimitriados & Pavlou Mela Street, GR 38333, Volos, Greece; Laboratory of Biochemistry, Faculty of Veterinary Science, University of Thessaly, GR-43100 Karditsa, Greece. Electronic address: gkontopidis@vet.uth.gr.
authors.forename: George
authors.initials: G
authors.lastname: Kontopidis
mesh_terms: D056751:Cyclin A2 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
chemical_list: D056751:Cyclin A2; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins
keywords: Molecular chaperones; Protein–ligand binding affinity; Recombinant human cyclin A2; Soluble expression; Tryptophan fluorescence titration