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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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

McKinstry WJ,Hijnen M,Tanwar HS,Sparrow LG,Nagarajan S,Pham ST,Mak J

doi

10.1016/j.pep.2014.04.013

subject

Has Abstract

pub_date

2014-08-01 00:00:00

pages

10-8

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(14)00091-6

journal_volume

100

pub_type

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