title: Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli.
abstract: The HIV-1 Gag precursor protein, Pr55(Gag), is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55(Gag) assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55(Gag) has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55(Gag) 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 Pr55(Gag) 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 Pr55(Gag) using a bacterial heterologous expression system. Recombinant HIV-1 Pr55(Gag) 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 Pr55(Gag) 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.
authors.affiliation: CSIRO Materials Science and Engineering, Parkville, Victoria, Australia.
authors.forename: William J
authors.initials: WJ
authors.lastname: McKinstry
authors.affiliation: Centre for Virology, Burnet Institute, Melbourne, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.
authors.forename: Marcel
authors.initials: M
authors.lastname: Hijnen
authors.affiliation: CSIRO Materials Science and Engineering, Parkville, Victoria, Australia; School of Medicine, Deakin University, Geelong, Australia.
authors.forename: Hanumant S
authors.initials: HS
authors.lastname: Tanwar
authors.affiliation: CSIRO Materials Science and Engineering, Parkville, Victoria, Australia.
authors.forename: Lindsay G
authors.initials: LG
authors.lastname: Sparrow
authors.affiliation: School of Medicine, Deakin University, Geelong, Australia; CSIRO, Australian Animal Health Laboratory, Geelong, Australia.
authors.forename: Sureshbabu
authors.initials: S
authors.lastname: Nagarajan
authors.affiliation: School of Medicine, Deakin University, Geelong, Australia; CSIRO, Australian Animal Health Laboratory, Geelong, Australia.
authors.forename: Son T
authors.initials: ST
authors.lastname: Pham
authors.affiliation: Centre for Virology, Burnet Institute, Melbourne, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia; School of Medicine, Deakin University, Geelong, Australia; CSIRO, Australian Animal Health Laboratory, Geelong, Australia. Electronic address: johnson.mak@deakin.edu.au.
authors.forename: Johnson
authors.initials: J
authors.lastname: Mak
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D012995:Solubility; D014169:Transformation, Bacterial
chemical_list: D008670:Metals; D011498:Protein Precursors; D011994:Recombinant Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1
keywords: Gag; HIV-1; Pr55(Gag); Purification; Recombinant protein expression