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Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli

McKinstry,WJ, Hijnen,M, Tanwar,HS, Sparrow,LG, Nagarajan,S, Pham,ST and Mak,J 2014, Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli, Protein expression and purification, vol. 100, pp. 10-18, doi: 10.1016/j.pep.2014.04.013.

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Title Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli
Author(s) McKinstry,WJ
Hijnen,M
Tanwar,HS
Sparrow,LG
Nagarajan,S
Pham,ST
Mak,JORCID iD for Mak,J orcid.org/0000-0002-5229-5707
Journal name Protein expression and purification
Volume number 100
Start page 10
End page 18
Total pages 9
Publisher Elsevier
Place of publication Amsterdam, The Netherlands
Publication date 2014-08
ISSN 1096-0279
Keyword(s) Gag
HIV-1
Pr55(Gag)
Purification
Recombinant protein expression
Amino Acid Sequence
Base Sequence
Chromatography, Affinity
Escherichia coli
Gene Expression
HIV Infections
Humans
Metals
Molecular Sequence Data
Plasmids
Protein Precursors
Recombinant Proteins
Solubility
Transformation, Bacterial
Science & Technology
Life Sciences & Biomedicine
Biochemical Research Methods
Biochemistry & Molecular Biology
Biotechnology & Applied Microbiology
DEFICIENCY SYNDROME AIDS
TYPE-1 MATRIX PROTEIN
VIRUS-LIKE PARTICLES
IN-VITRO
GAG PROTEIN
CAPSID PROTEIN
ASSEMBLY PROPERTIES
LIPID RAFTS
P6 DOMAIN
IMMUNODEFICIENCY
Summary 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.
Language eng
DOI 10.1016/j.pep.2014.04.013
Field of Research 110399 Clinical Sciences not elsewhere classified
Socio Economic Objective 929999 Health not elsewhere classified
HERDC Research category C1 Refereed article in a scholarly journal
ERA Research output type C Journal article
Copyright notice ©2014, Elsevier
Persistent URL http://hdl.handle.net/10536/DRO/DU:30070387

Document type: Journal Article
Collection: School of Medicine
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