title: Bovine Sex Determining Region Y: Cloning, Optimized Expression, and Purification.
abstract: Sex determining region Y gene (SRY) is located on Y chromosome and encodes a protein with 229 amino acids. In this study, ORF region of SRY with a length of 690 bp was synthesized using PCR and ligated to pET28a (+), then transformed in E.coli DH5α. E.coli BL21 (DE3) strain was chosen to express recombinant bovine SRY protein. A set of optimization steps was taken including different concentrations of IPTG, glucose, and temperatures at differed incubation times after the induction. Results showed that temperature points and different concentrations of IPTG and glucose had a significant effect (p < 0.01) on total protein and recombinant bovine SRY. After purification, various temperatures and concentrations of IPTG showed meaningful effects (p < 0.01) on the solubility of expressed recombinant SRY. Highest soluble rSRY protein amount was achieved where 0.5 mM IPTG and 0.5% glucose was used at 20°C during induction. In the absence of glucose, the highest amount of soluble recombinant SRY levels were achieved at the concentrations of 0.8 mM of IPTG at 28°C, 20°C, and 1.5 mM IPTG at 37°C during induction for 16, 24, and 8 hours, respectively. Regarding the results obtained in this study, it could be stated that by decreasing temperature and inducer concentration, soluble bovine SRY protein expression increases.
authors.affiliation: a Medical Biology Research Center , Kermanshah University of Medical Sciences , Kermanshah , Iran.|b Department of Animal Science, Faculty of Animal and Fishery Sciences , Agricultural Science and Natural Resources University of Sari , Sari , Iran.
authors.forename: Bijan
authors.initials: B
authors.lastname: Soleymani
authors.affiliation: b Department of Animal Science, Faculty of Animal and Fishery Sciences , Agricultural Science and Natural Resources University of Sari , Sari , Iran.
authors.forename: Sayed Hassan
authors.initials: SH
authors.lastname: Hafezian
authors.affiliation: b Department of Animal Science, Faculty of Animal and Fishery Sciences , Agricultural Science and Natural Resources University of Sari , Sari , Iran.
authors.forename: Ghodratollah Rahimi
authors.initials: GR
authors.lastname: Mianji
authors.affiliation: a Medical Biology Research Center , Kermanshah University of Medical Sciences , Kermanshah , Iran.
authors.forename: Kamran
authors.initials: K
authors.lastname: Mansouri
authors.affiliation: c Agricultural Research Center , Jehad-e Keshavarzi Institute , Kermanshah , Iran.
authors.forename: Broomand
authors.initials: B
authors.lastname: Chaharaein
authors.affiliation: a Medical Biology Research Center , Kermanshah University of Medical Sciences , Kermanshah , Iran.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Tajehmiri
authors.affiliation: d Schools of Molecular Bioscience , University of Sydney , Sydney , New South Wales , Australia.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Sharifi Tabar
authors.affiliation: a Medical Biology Research Center , Kermanshah University of Medical Sciences , Kermanshah , Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Mostafaie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D024282:Genes, sry / Q000235:genetics*; D008297:Male; D016366:Open Reading Frames / Q000235:genetics; D000072417:Protein Domains; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA / Q000662:veterinary; D051876:Sex-Determining Region Y Protein / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D014998:Y Chromosome / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D051876:Sex-Determining Region Y Protein
keywords: Cloning; expression; purification; recombinant bovine SRY protein; sex determinant region Y chromosome