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Introduction: Background: Neff regulatory protein as well as structural proteins such as MPER and V3 of HIV-1 is important targets in vaccine studies. Therefore, in the current study, the Nef-MPER-V3 fusion protein was cloned and expressed in prokaryotic expression system that will be evaluated as a candidate for future vaccine studies. Materials and Methods: The Nef-MPER-V3 sequence was synthesized and inserted into pET-28a(+) vector using Not I/HindIII enzymes. Then, the recombinant construct was expressed in BL21 (DE3) strain of E.coli. For optimization of protein expression, several factors such as time of induction, IPTG concentration and optical density (OD) were evaluated. The recombinant protein was purified on a Ni-NTA agarose column and analyzed by 12% SDS-PAGE. Finally, the fusion protein was confirmed by western blotting. Results: The target gene was successfully cloned into the recombinant vector. The best condition for the expression such as; 2 m IPTG, OD600 = 0.7 and growth 5 hours post-induction were determined and the recombinant protein was purified by affinity chromatography in denaturing condition with urea at a concentration of about 300 μg/ ml. The purified fusion protein migrated as a clear band of around 35 kDa in SDS-PAGE and was detectable using anti-Neff antibody in western blotting. Conclusion: Our results showed that the Nef-MPER-V3 protein was successfully expressed in prokaryotic system and purified protein may provide the antigen for future experiments for immunogenicity evaluation in mice is currently undertaken. [ABSTRACT FROM AUTHOR]