Effects of Disruption of the Nucleotide Pattern in Crid Element and Kozak Sequence of Interferon Β on Mrna Stability and Protein Production Publisher Pubmed



Kay M¹ ; Hojati Z² ; Heidari M² ; Bazi Z³ ; Korbekandi H⁴ Show Affiliations
Source: Autoimmunity Published:2015

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 IFNβK and IFNβK+CRID were designed by site-directed mutagenesis. The IFNβK and IFNβK+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 IFNβK has been elevated up to 3.5-fold. On the other hand, enormous amounts of IFNβ mRNA and protein were produced by cells containing IFNβK+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. © 2015 © 2015 Informa UK Ltd. All rights reserved: reproduction in whole or part not permitted.