Effects of disruption of the nucleotide pattern in CRID element and Kozak sequence of interferon beta on mRNA stability and protein production

Abstract

Interferon beta (IFN beta) is the most important drug that has been used frequently for multiple sclerosis treatment. This study has tried to improve the IFN beta production by introducing mutations in the coding region of IFN beta, while its amino acid sequence is intact. Two recombinant vectors IFN beta(K) and IFN beta(K+CRID) were designed by site-directed mutagenesis. The IFN beta(K) and IFN beta(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 IFN beta protein produced by CHO cells containing IFN beta(K) has been elevated up to 3.5-fold. On the other hand, enormous amounts of IFN beta mRNA and protein were produced by cells containing IFN beta(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 IFN beta mRNA stability and, may also enhance mRNA half-life. In a similar way, more proteins are produced by modification of Kozak sequence.