Expression and purification of soluble recombinant full length HIV-1 Pr55Gag protein in Escherichia coli

The HIV-1 Gag precursor protein, Pr55Gag, is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55Gag assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55Gag has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55Gag for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag's natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55Gag to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55Gag using a bacterial heterologous expression system. Recombinant HIV-1 Pr55Gag was expressed with a C-terminal His×6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55Gag that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.