İnsan beta interferon geninin klonlanması ve E.coli'de ekspresyonu üzerine moleküler çalışmalar

Human interferon beta (hIFNβ), produced by many cell types in mammals in response to viral and nonviral infections, acts as an immunoregulator and have a potential to become an important therapeutic drug for several diseases such as multiple sclerosis, asthma, hepatitis B and C, human papillomavirus and various cancers. Considering the clinical applications and convenience of the rIFNβ production due to its molecular size, generating a low-cost production system, which could provide as much as pure and high yield of this therapeutic protein, is very important. Prokaryotic secretory systems are mostly preferred for the production in order to secrete proteins into the periplasm or out of the cell membrane. Thus, secretoy system simplifies the downstream processing and reduces the protein degradation and cost. It is a quietly desirable and customisable system for secretion of IFNβ1b. In Turkey, licensed domestic production of recombinant proteins, imported at high prices, has not been achieved. There are some reports describing the construction of novel vectors expressing human rIFNβ1b in the periplasm of the E. coli cells. In this study, for production of rIFNβ1b protein in periplasmic space, codon optimisated rIFNβ1b gene according to E. coli codon table was provided by in silico bioinformatic tools. The obtained gene was cloned into pET22b expression vector which contains the pelB signal peptide. Then recombinant IFNβ1b peptide synthesis was performed in E. coli/BLR(DE)3 strain induced with 0.4 mM IPTG at 25°C and then isolated from the medium, periplasmic and cytoplasmic space of the host and they were pufied with the Ni-NTA affinity colon. The production of the rIFNβ1b, (~18 kDa) was determined by %12 SDS-PAGE and Western Blot analysis. Our results indicated that the intracellular soluble expression of the optimized IFNβ1b protein did not take place. Eventhough, this was not confirmed by experimetally, we thought that because of the improper codon optimization, folded form of rIFNβ1b mRNA was produced and this structure prevented the translation of rIFNβ1b mRNA. Reconstruction of the prokaryotic secretion system and codon optimizations may improve the more convenient production and purification of rIFNβ1b protein.