title: Targeted mutations in Val101 and Arg27 interferon beta protein increase its transcriptional and translational activities.
abstract: Interferon β (IFNβ) is the most prescribed drug that has been used frequently for the treatment of multiple sclerosis (MS) patients. The aim of this study is to improve the production of IFNβ by induction of site directed mutagenesis. Accordingly, recombinant constructs were designed in order to enhance the expression of IFNβ mRNA and protein. The recombinant plasmids were transfected to the CHO cell line, following RNA extractions and cDNA synthesis. The effects of recombinant constructs were analyzed by real time PCR, ELISA and MTT assay. Transfected samples with either IFNβ101 or IFNβ101+27 have shown 11.55 and 2.26 fold elevation and over-expression compare to the wild type construct respectively. Our data also indicated that the IFNβ101 and IFNβ101+27 constructs increase IFNβ protein expression more than 2.2 and 4.5 fold, respectively compared to the control group. It could be concluded that the substitution of Phe in the codon 101 position, which may increase the binding activity of IFNβ with its receptors and introduction of an additional N glycosylation site (Asn-X-Thr) in the position 27 of IFNβ protein may cause such an effect. The proliferative activity of transfected cells by a recombinant IFNβ101 decreases in comparison to the wild type, although it was not statistically significant. Over-expression of IFNβ in such a level is promising not only for the patients but also for the pharmaceutical industries.
authors.affiliation: Department of Molecular Genetics, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Kay
authors.affiliation: Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Amin Moradi
authors.initials: AM
authors.lastname: Hasan-Abad
authors.affiliation: Division of Genetics, Biology Department, Faculty of Sciences, University of Isfahan, Isfahan, Iran. Electronic address: z.hojati@sci.ui.ac.ir.
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Hojati
authors.affiliation: Genetics & Molecular Biology Department, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Hasan
authors.initials: H
authors.lastname: Korbekandi
mesh_terms: D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D003062:Codon / Q000235:genetics; D003412:Cricetulus; D005786:Gene Expression Regulation; D006801:Humans; D016899:Interferon-beta / Q000096:biosynthesis* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D014176:Protein Biosynthesis; D060888:Real-Time Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D003062:Codon; D016899:Interferon-beta
keywords: ARE elements; Codon 101; Interferon beta; Real-time PCR