Large-scale functional purification of recombinant HIV-1 capsid.

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 infectivit...

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Main Authors: Magdeleine Hung, Anita Niedziela-Majka, Debi Jin, Melanie Wong, Stephanie Leavitt, Katherine M Brendza, Xiaohong Liu, Roman Sakowicz
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2013-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC3589475?pdf=render
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spelling doaj-0dc931a917b94da785019ee58099729c2020-11-25T00:43:23ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0183e5803510.1371/journal.pone.0058035Large-scale functional purification of recombinant HIV-1 capsid.Magdeleine HungAnita Niedziela-MajkaDebi JinMelanie WongStephanie LeavittKatherine M BrendzaXiaohong LiuRoman SakowiczDuring 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.http://europepmc.org/articles/PMC3589475?pdf=render
collection DOAJ
language English
format Article
sources DOAJ
author Magdeleine Hung
Anita Niedziela-Majka
Debi Jin
Melanie Wong
Stephanie Leavitt
Katherine M Brendza
Xiaohong Liu
Roman Sakowicz
spellingShingle Magdeleine Hung
Anita Niedziela-Majka
Debi Jin
Melanie Wong
Stephanie Leavitt
Katherine M Brendza
Xiaohong Liu
Roman Sakowicz
Large-scale functional purification of recombinant HIV-1 capsid.
PLoS ONE
author_facet Magdeleine Hung
Anita Niedziela-Majka
Debi Jin
Melanie Wong
Stephanie Leavitt
Katherine M Brendza
Xiaohong Liu
Roman Sakowicz
author_sort Magdeleine Hung
title Large-scale functional purification of recombinant HIV-1 capsid.
title_short Large-scale functional purification of recombinant HIV-1 capsid.
title_full Large-scale functional purification of recombinant HIV-1 capsid.
title_fullStr Large-scale functional purification of recombinant HIV-1 capsid.
title_full_unstemmed Large-scale functional purification of recombinant HIV-1 capsid.
title_sort large-scale functional purification of recombinant hiv-1 capsid.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2013-01-01
description 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.
url http://europepmc.org/articles/PMC3589475?pdf=render
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