title: mRNA secondary structure modulates the translation of organophosphate hydrolase (OPH) in E. coli.
abstract: Organophosphate hydrolases (OPHs), involved in hydrolytic cleavage of structurally diverse organophosphates are coded by a plasmid borne, highly conserved organophosphate degrading (opd) gene. An inverted repeat sequence found in the signal coding region of the opd gene was found to be responsible for inducing a stable stem loop structure with a DeltaG of -23.1 kcal/mol. This stem loop structure has shown significant influence on the expression levels of organophosphate hydrolase (OPH) in E. coli. When the signal coding region comprising the inverted repeat sequence was deleted a approximately 3.28 fold increase in the expression levels of OPH was noticed in E. coli BL21 cells. Mutations in the inverted repeat region, especially at the third position of the codon, to a non-complementary base destabilized the secondary structure of opd mRNA. When such opd variant, opd' was expressed, the expression levels were found to be similar to expression levels coded by the construct generated by deleting the signal peptide coding region. Deletion of signal peptide did not influence the folding and activity of OPH. Though high level induction has resulted in accumulation of OPH as inclusion bodies, modulation of expression levels by reducing the copy number of the expression plasmid, inducer concentration and growth temperature has produced majority of the protein in soluble and active form.
authors.affiliation: Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India.
authors.forename: Jay Prakash
authors.initials: JP
authors.lastname: Pandey
authors.forename: Purushotham
authors.initials: P
authors.lastname: Gorla
authors.forename: Bramanandam
authors.initials: B
authors.lastname: Manavathi
authors.forename: Dayananda
authors.initials: D
authors.lastname: Siddavattam
mesh_terms: D001483:Base Sequence; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D009690:Nucleic Acid Conformation; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000235:genetics*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics
chemical_list: D012329:RNA, Bacterial; D012333:RNA, Messenger; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases