title: A non-cleavable hexahistidine affinity tag at the carboxyl-terminus of the HIV-1 Pr55Gagpolyprotein alters nucleic acid binding properties.
abstract: HIV Gag (Pr55Gag), a multidomain polyprotein that orchestrates the assembly and release of the human immunodeficiency virus (HIV), is an active target of antiretroviral inhibitor development. However, highly pure, stable, recombinant Pr55Gaghas been difficult to produce in quantities sufficient for biophysical studies due to its susceptibility to proteolysis by cellular proteases during purification. Stability has been improved by using a construct that omits the p6 domain (Δp6). In vivo, p6 is crucial to the budding process and interacts with protein complexes in the ESCRT (Endosomal Sorting Complexes Required for Transport) pathway, it has been difficult to study its role in the context of Gag using in vitro approaches. Here we report the generation of a full length Gag construct containing a tobacco etch virus (TEV)-cleavable C-terminal hexahistidine tag, allowing a detailed comparison of its nucleic acid binding properties with other constructs, including untagged, Δp6, and C-terminally tagged (TEV-cleavable and non-cleavable) Gags, respectively. We have developed a standard expression and purification protocol that minimizes nucleic acid contamination and produces milligram quantities of full length Gag for in vitro studies and compound screening purposes. We found that the presence of a carboxyl-terminal hexahistidine tag changes the nucleic binding properties compared to the proteins that did not contain the tag (full length protein that was either untagged or reulted from removal of the tag during purification). The HIV Gag expression and purification protocol described herein provides a facile method of obtaining large quantities of high quality protein for investigators who wish to study the full length protein or the effect of the p6 domain on the biophysical properties of Gag.
authors.affiliation: Department of Biochemistry and Molecular Biology, Penn State College of Medicine, 500 University Drive, Hershey, PA, 17033, USA.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Bewley
authors.affiliation: Department of Biochemistry and Molecular Biology, Penn State College of Medicine, 500 University Drive, Hershey, PA, 17033, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Reinhart
authors.affiliation: Department of Microbiology and Immunology, Penn State College of Medicine, 500 University Drive, Hershey, PA, 17033, USA; Department of Medicine, Penn State College of Medicine, 500 University Drive, Hershey, PA, 17033, USA.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Stake
authors.affiliation: Department of Microbiology and Immunology, Penn State College of Medicine, 500 University Drive, Hershey, PA, 17033, USA; Department of Medicine, Penn State College of Medicine, 500 University Drive, Hershey, PA, 17033, USA.
authors.forename: Shorena
authors.initials: S
authors.lastname: Nadaraia-Hoke
authors.affiliation: Department of Microbiology and Immunology, Penn State College of Medicine, 500 University Drive, Hershey, PA, 17033, USA; Department of Medicine, Penn State College of Medicine, 500 University Drive, Hershey, PA, 17033, USA.
authors.forename: Leslie J
authors.initials: LJ
authors.lastname: Parent
authors.affiliation: Department of Biochemistry and Molecular Biology, Penn State College of Medicine, 500 University Drive, Hershey, PA, 17033, USA. Electronic address: jmf27@psu.edu.
authors.forename: John M
authors.initials: JM
authors.lastname: Flanagan
mesh_terms: D004247:DNA / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D006639:Histidine / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D011485:Protein Binding; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; C033223:polyhistidine; D006639:Histidine; D004247:DNA
keywords: Gag; HIV; His-tag; Nucleic acid binding; Pr55(Gag); Proteolysis; p6