Evaluating secretory expression of human interferon alpha-2a in E. coli using selective signal peptide from in silico studies

Abstract

Abstract Introduction The secretory expression of recombinant proteins has many advantages, which has attracted the attention of scientists for the secretory production of heterologous proteins. A critical element in the effective secretion of recombinant proteins is the signal peptide (SP). IFN-alpha-2a is a multifunctional cytokine that is being used for HCV-infected patients, Kaposi’s sarcoma, melanomas, and chronic myelogenous leukemia (CML). Aim: This study was aimed at in silico evaluation of different signal peptides to identify the most efficient signal peptides for secretory production of the IFN-alpha-2a in E. coli. Method: Firstly, “SignalP 4.1” was employed to predict SPs as well as their cleavage site. Accordingly, five SPs, including OSPA1_BORBU, CDGT_BACS2, CPLR_DESHA, SPA_STAAU, and LPP_E. coli were excluded from further analysis since they had cleavage problems. Various physicochemical features of signal peptides with the potential to affect protein secretion were examined by ProtParam and SOL pro servers. Subcellular localization sites and secretion sorting were studied by ProtcompB and PRED-TAT software programs. For in vitro analysis, the coding region for IFN-alpha-2a was amplified by PCR using specific primers. The amplicon was then ligated into the pET26b expression vector, transformed into the E.coli BL21 and protein expression was analyzed by SDS-PAGE (in different IPTG concentrations and temperatures) Results and conclusion: This analysis revealed that Chaperone protein sfmC (SFMC_ECOLI), Acid shock protein (ASR_ECOLI), Protease do (DEGP_ECOLI), and Thiol: disulfide interchange protein dsbA (DSBA_ECOLI) could be considered as efficient SPs for secretory expression of IFN-alpha-2a in E. coli.. Agarose gel electrophoresis of PCR amplification product of target gene showed a 600 bp fragment that was consistent with the expected size of IFN-alpha-2a+ SFMC_ECOLI. Sequencing of cloned fragment confirmed the identity of the product as IFN-alpha-2a. Expression of human recombinant protein IFN-alpha-2a in E. coli BL21 produced high levels of recombinant protein that appeared as a dense band in SDS-PAGE analysis. The results showed that the majority of recombinant protein is expressed in periplasmic space.