title: Effects of disruption of the nucleotide pattern in CRID element and Kozak sequence of interferon β on mRNA stability and protein production.
abstract: Interferon β (IFNβ) is the most important drug that has been used frequently for multiple sclerosis treatment. This study has tried to improve the IFNβ production by introducing mutations in the coding region of IFNβ, while its amino acid sequence is intact. Two recombinant vectors IFNβ(K) and IFNβ(K+CRID )were designed by site-directed mutagenesis. The IFNβ(K) and IFNβ(K+CRID) have two substitutions in Kozak sequence and four substitutions in CRID sequence, respectively. The Chinese hamster ovary (CHO) cell codon usage optimization was also performed for both of them. They were transiently transfected to CHO-dhfr(-) cell line using Lipofectamine kit (Invitrogen, Grand Island, NY). The amount of mRNA and protein was determined by real time PCR and ELISA. The results of this study indicate that the amount of IFNβ protein produced by CHO cells containing IFNβ(K) has been elevated up to 3.5-fold. On the other hand, enormous amounts of IFNβ mRNA and protein were produced by cells containing IFNβ(K+CRID) construct; more than 4.6-fold and 6-fold, respectively. It could be concluded that disruption of AT pattern in CRID element increase RNA and protein production, improve IFNβ mRNA stability and, may also enhance mRNA half-life. In a similar way, more proteins are produced by modification of Kozak sequence.
authors: Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University , Tehran , Iran .
authors: Maryam
authors: M
authors: Kay
authors: Zohreh
authors: Z
authors: Hojati
authors: Maryam
authors: M
authors: Heidari
authors: Zahra
authors: Z
authors: Bazi
authors: Hassan
authors: H
authors: Korbekandi
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003412:Cricetulus; D015870:Gene Expression; D016899:Interferon-beta / Q000096:biosynthesis* / Q000235:genetics*; D059372:Nucleotide Motifs; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D020871:RNA Stability; D020218:Response Elements
chemical_list: D020413:3' Untranslated Regions; D016899:Interferon-beta
keywords: CRID sequence; ELISA; Kozak sequence; interferon β; real time PCR