EMAIL THIS PAGE TO A FRIEND

PloS one

Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion.


PMID 26295457

Abstract

The membrane proximal region (MPR, residues 649-683) and transmembrane domain (TMD, residues 684-705) of the gp41 subunit of HIV-1's envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662-683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649-705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.

Related Materials

Product #

Image

Description

Molecular Formula

Add to Cart

B4429
BIS-TRIS, BioPerformance Certified, cell culture tested, suitable for insect cell culture, ≥98.0%
C8H19NO5
B9754
BIS-TRIS, ≥98.0% (titration)
C8H19NO5
RES1161B-A7
BIS-TRIS, PharmaGrade, Manufactured under appropriate controls for use as a raw material in pharma or biopharmaceutical production., suitable for cell culture
C8H19NO5
PHG0004
BIS-TRIS, PharmaGrade, Manufactured under appropriate controls for use as a raw material in pharma or biopharmaceutical production, suitable for cell culture
C8H19NO5
B7535
BIS-TRIS, BioXtra, ≥98.0% (titration)
C8H19NO5
14879
BIS-TRIS, BioUltra, ≥99.0% (NT)
C8H19NO5
N9877
Nitrilotriacetic acid, Sigma Grade, ≥99%
C6H9NO6
72560
Nitrilotriacetic acid, ACS reagent, ≥99.0%
C6H9NO6
72559
Nitrilotriacetic acid, BioUltra, ≥99.0% (T)
C6H9NO6
P5655
Potassium phosphate monobasic, powder, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
H2KO4P
RES20760-A7
Potassium phosphate monobasic, PharmaGrade, NF, Manufactured under appropriate GMP controls for pharma or biopharmaceutical production.
H2KO4P
P8416
Potassium phosphate monobasic, plant cell culture tested, ≥99%
H2KO4P
P9791
Potassium phosphate monobasic, for molecular biology, ≥98.0%
H2KO4P
229806
Potassium phosphate monobasic, 99.99% trace metals basis
H2KO4P
P5379
Potassium phosphate monobasic, ReagentPlus®
H2KO4P
60218
Potassium phosphate monobasic, BioUltra, for molecular biology, anhydrous, ≥99.5% (T)
H2KO4P
S8830
SIGMAFAST Protease Inhibitor Cocktail Tablets, EDTA-Free, for use in purification of Histidine-tagged proteins