Expression and purification of soluble recombinant full length HIV-1 Pr55Gag protein in Escherichia coli

McKinstry, William J.; Hijnen, Marcel; Tanwar, Hanumant S.; Sparrow, Lindsay G.; Nagarajan, Sureshbabu; Pham, Son Bao; Mak, Johnson

doi:10.1016/j.pep.2014.04.013

Cited by 27 publications

(43 citation statements)

References 41 publications

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“…Large amounts of Pr55 Gag were produced in Escherichia coli and purified to homogeneity in two high-performance liquid chromatography steps 47 . Suboptimal expression conditions limited incorporation into inclusion bodies and proteolysis, and addition of a C-terminal His 6 -tag allowed efficient separation of the proteolytic cleavage products from the intact protein ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

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Specific recognition of the HIV-1 genomic RNA by the Gag precursor

et al. 2014

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Section: Resultsmentioning

confidence: 99%

“…Expression, purification and characterization of NL4.3 Pr55 Gag and GagDp6 with an appended C-terminal His 6 -tag were performed as recently described 47 . The purity and identity of all protein preparations was confirmed using SDS-PAGE (Supplementary Fig.…”

Section: Methodsmentioning

confidence: 99%

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

et al. 2014

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“…Recombinant Gag proteins (Pr55 Gag , Pr50 Δp6Gag , Pr55 Gag WM 316–7 AA , Pr55 Gag [CA Helix 6 TTSTLQ 239–44 AASALA], Pr55 Gag [CA Helix 10 D329A], Pr55 Gag [CA All 4]) and processed NC proteins (p15 NC-SP2-p6 , p7 NC ) were expressed with C-term His-tag and purified as previously described [45]. Large scale production and purification of Pr55 Gag is described herein.…”

Section: Methodsmentioning

confidence: 99%

The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV

et al. 2017

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The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly.

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“…In addition, McKinstry et al . reported the development of a bacterial heterologous protein expression system to produce soluble recombinant wild type full length HIV-1 Pr55 Gag using a C-terminal hexahistidine tag (Pr55 Gag -His) to facilitate purification [4]. Although this approach was an important step forward and improved the purity of the protein, the possible effect of a non-cleavable C-terminal His tag on the function of the p6 domain was not examined.…”

Section: Introductionmentioning

confidence: 99%

A non-cleavable hexahistidine affinity tag at the carboxyl-terminus of the HIV-1 Pr55Gag polyprotein alters nucleic acid binding properties

Bewley

Reinhart

Stake

et al. 2017

Protein Expression and Purification

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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 Pr55Gag has 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.

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Expression and purification of soluble recombinant full length HIV-1 Pr55Gag protein in Escherichia coli

Cited by 27 publications

References 41 publications

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV

A non-cleavable hexahistidine affinity tag at the carboxyl-terminus of the HIV-1 Pr55Gag polyprotein alters nucleic acid binding properties

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