Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41

Zhen Gong, Sarah A. Kessans, Lusheng Song, Katerina Dörner, Ho Hsien Lee, Lydia R. Meador, Joshua Labaer, Brenda G. Hogue, Tsafrir S. Mor, Petra Fromme

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the post-fusion forms. Structural information on the TM domain of gp41 is scant and at low resolution. Here we describe the design, expression and purification of a protein construct that includes MPR and the transmembrane domain of gp41 (MPR-TMTEV-6His), which reacts with the broadly neutralizing antibodies 2F5 and 4E10 and thereby may represent an immunologically relevant conformation mimicking a prehairpin intermediate of gp41. The expression level of MPR-TMTEV-6His was improved by fusion to the C-terminus of Mistic protein, yielding ∼1 mg of pure protein per liter. The isolated MPR-TMTEV-6His protein was biophysically characterized and is a monodisperse candidate for crystallization. This work will enable further investigation into the structure of MPR-TMTEV-6His, which will be important for the structure-based design of a mucosal vaccine against HIV-1.

Original languageEnglish
Pages (from-to)1607-1618
Number of pages12
JournalProtein Science
Volume23
Issue number11
DOIs
Publication statusPublished - Nov 1 2014

Fingerprint

Purification
HIV-1
Membranes
Neutralizing Antibodies
Glycoproteins
Proteins
Fusion reactions
Infectious Disease Transmission
Crystallization
Anchors
Conformations
Epitopes
Vaccines
X-Rays
X rays

Keywords

  • dynamic light scattering
  • gp41
  • HIV-1
  • membrane proximal region
  • surface plasmon resonance
  • transmembrane domain

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41. / Gong, Zhen; Kessans, Sarah A.; Song, Lusheng; Dörner, Katerina; Lee, Ho Hsien; Meador, Lydia R.; Labaer, Joshua; Hogue, Brenda G.; Mor, Tsafrir S.; Fromme, Petra.

In: Protein Science, Vol. 23, No. 11, 01.11.2014, p. 1607-1618.

Research output: Contribution to journalArticle

Gong, Z, Kessans, SA, Song, L, Dörner, K, Lee, HH, Meador, LR, Labaer, J, Hogue, BG, Mor, TS & Fromme, P 2014, 'Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41', Protein Science, vol. 23, no. 11, pp. 1607-1618. https://doi.org/10.1002/pro.2540
Gong, Zhen ; Kessans, Sarah A. ; Song, Lusheng ; Dörner, Katerina ; Lee, Ho Hsien ; Meador, Lydia R. ; Labaer, Joshua ; Hogue, Brenda G. ; Mor, Tsafrir S. ; Fromme, Petra. / Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41. In: Protein Science. 2014 ; Vol. 23, No. 11. pp. 1607-1618.
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