title: Possible Function of the ribT Gene of Bacillus subtilis: Theoretical Prediction, Cloning, and Expression.
abstract: The complete decipherment of the functions and interactions of the elements of the riboflavin biosynthesis operon (rib operon) of Bacillus subtilis are necessary for the development of superproducers of this important vitamin. The function of its terminal ribT gene has not been established to date. In this work, a search for homologs of the hypothetical amino acid sequence of the gene product through databases, as well as an analysis of the homolgs, was performed; the distribution of secondary structure elements was theoretically predicted; and the tertiary structure of the RibT protein was proposed. The ribT gene nucleotide sequence was amplified and cloned into the standard high-copy expression vector pET15b and then expressed after induction with IPTG in E. coli BL21 (DE3) strain cells containing the inducible phage T7 RNA polymerase gene. The ribT gene expression was confirmed by SDS-PAGE. The protein product of the expression was purified by affinity chromatography. Therefore, the real possibility of RibT protein production in quantities sufficient for further investigation of its structure and functional activity was demonstrated.
authors.affiliation: B.P. Konstantinov Petersburg Nuclear Physics Institute, National Research Center "Kurchatov Institute", Orlova Roshcha, Gatchina, Leningrad Region, Russia, 188300 ; St. Petersburg State Polytechnical University, Polytechnicheskaya Str., 29, St. Petersburg, Russia, 195251.
authors.forename: A P
authors.initials: AP
authors.lastname: Yakimov
authors.affiliation: State Research Institute of Genetics and Selection of Industrial Microorganisms, 1st Dorozhnyi Proezd, 1, Moscow, Russia, 117545.
authors.forename: T A
authors.initials: TA
authors.lastname: Seregina
authors.affiliation: State Research Institute of Genetics and Selection of Industrial Microorganisms, 1st Dorozhnyi Proezd, 1, Moscow, Russia, 117545.
authors.forename: A A
authors.initials: AA
authors.lastname: Kholodnyak
authors.affiliation: B.P. Konstantinov Petersburg Nuclear Physics Institute, National Research Center "Kurchatov Institute", Orlova Roshcha, Gatchina, Leningrad Region, Russia, 188300.
authors.forename: R A
authors.initials: RA
authors.lastname: Kreneva
authors.affiliation: State Research Institute of Genetics and Selection of Industrial Microorganisms, 1st Dorozhnyi Proezd, 1, Moscow, Russia, 117545.
authors.forename: A S
authors.initials: AS
authors.lastname: Mironov
authors.affiliation: B.P. Konstantinov Petersburg Nuclear Physics Institute, National Research Center "Kurchatov Institute", Orlova Roshcha, Gatchina, Leningrad Region, Russia, 188300.
authors.forename: D A
authors.initials: DA
authors.lastname: Perumov
authors.affiliation: B.P. Konstantinov Petersburg Nuclear Physics Institute, National Research Center "Kurchatov Institute", Orlova Roshcha, Gatchina, Leningrad Region, Russia, 188300 ; St. Petersburg State Polytechnical University, Polytechnicheskaya Str., 29, St. Petersburg, Russia, 195251.
authors.forename: A L
authors.initials: AL
authors.lastname: Timkovskii
keywords: bioinformatics; gene cloning; homology search; inducible expression; proteomics; theoretical protein structure