title: Large-scale functional purification of recombinant HIV-1 capsid.
abstract: During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.
authors.affiliation: Gilead Sciences Inc., Foster City, California, USA.
authors.forename: Magdeleine
authors.initials: M
authors.lastname: Hung
authors.forename: Anita
authors.initials: A
authors.lastname: Niedziela-Majka
authors.forename: Debi
authors.initials: D
authors.lastname: Jin
authors.forename: Melanie
authors.initials: M
authors.lastname: Wong
authors.forename: Stephanie
authors.initials: S
authors.lastname: Leavitt
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Brendza
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Liu
authors.forename: Roman
authors.initials: R
authors.lastname: Sakowicz
mesh_terms: D036022:Capsid Proteins / Q000302:isolation & purification*; D002845:Chromatography; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000378:metabolism; D015497:HIV-1 / Q000302:isolation & purification*; D013058:Mass Spectrometry; D046529:Microscopy, Electron, Transmission; D009154:Mutation; D011994:Recombinant Proteins / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance; D014461:Ultracentrifugation
chemical_list: D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins