Alireza Milani, Azam Bolhassani, Masoumeh Heshmati: Comparison of HIV-Nef protein expression in various E. coli expression systems to the purpose of vaccine design. The Second International and 10th National Biotechnology Congress, Tehran-Iran; 08/2017. (Poster)
Abstract: Protein vaccine has been known as one of the most effective vaccination strategies. HIV-1 nef gene, a regulatory gene, plays a critical role in viral life cycle and infection due to its multiple immunogenic epitopes, thus it has been considered as a suitable candidate for vaccine design. In this study, expression and purification of Nef protein was performed in different prokaryotic expression systems such as pQE30/M15 and pET23.a/Rosetta for evaluation of the best efficiency of these systems. The results of cloning in both systems confirmed the existence of a ~648 bp band related to nef gene on the agarose gel. Also, analysis of SDS-PAGE and Western Blot showed that expression of Nef protein in BL21 and Rosetta bacterial cells would be possible at 37 °C and 16 hours after induction by IPTG, whereas no expression observed for M15 system. Moreover, purification of Nef protein under both native and denaturing conditions using affinity chromatography indicated that the efficiency of denaturing approach is higher than native method. Regarding high expression of the Nef protein in host E. coli pET/Rosetta, this recombinant protein can be used as an antigen in the design of a protein vaccine against HIV-1 infection.
Keywords: Nef, HIV-1, cloning, expression and purification
