Cloning, expression and nanodiscs assemble of recombinant HIV-1 gp41. (January 2020)

• Record Type: Journal Article
• Authors: Elbahnasawy, Mostafa A.; Farag, Mohamed M.S.; Mansour, Mohamed T.; El-Ghamery, Abbas A.
• Is Part Of: Microbial pathogenesis. Volume 138(2020)
• Publication Date: 2020-01
• Subjects: HIV-1 gp41 MPER -- Cloning -- Expression -- Nanodiscs; HIV human immunodeficiency virus -- gp glycoprotein -- MPER membrane proximal external region -- GB1 B1 domain of Streptococcal protein G -- Trx thioredoxin -- bNAbs broadly neutralizing antibodies -- NDs nanodiscs; Pathogenic microorganisms -- Periodicals; Pathology, Molecular -- Periodicals; Communicable Diseases -- microbiology -- Periodicals; Communicable Diseases -- parasitology -- Periodicals; Micro-organismes pathogènes -- Périodiques; Pathologie moléculaire -- Périodiques; Electronic journals; 616.9041
• DOI: 10.1016/j.micpath.2019.103824
• Languages: English
• ISSNs: 0882-4010

Abstract

Abstract: Structural studies of membrane proteins have been hurdled by their difficulty for expression in heterogeneous expression systems due to their intrinsically strong hydrophobicity and requirements for association with other cellular membranes. This study aims to design a construct for expression of membrane proteins. Because of its outstanding interest in HIV-1 vaccine design, transmembrane gp41 amino acid residue 662–723 was chosen as a representative membrane protein. Therefore, we constructed expression vectors for expression of gp41(662-723) alone (pET28a-gp41(662-723) ) or coupled with a fusion partner: GB1 (pET30a-GB1-gp41(662-723) ) and Trx (pET32a-Trx-gp41(662-723) ). For enhancing protein expression, the expression plasmids were transformed into E. coli BL-21 (DE3), E. coli T7 Express lysY/I q and E. coli Lemo21 (DE3). Interestingly, HIV-1 gp41(662-723) was expressed as a C-terminus fusion to the fusion partner Trx (Trx-gp41(662-723) ) with an apparent molecular mass of 21.8 kDa. Trx-gp41(662-723) was overexpressed into E. coli T7 Express lysY/I q by early induction as OD600 ~0.5 followed by incubation at 20 °C/overnight. Our data demonstrated that almost all recombinant Trx-gp41(662-723) was incorporated into lipid nanodiscs by slowing down the nanodiscs assembly process. Negative-stained electron micrographs revealed homogenous 10 nm Trx-gp41(662-723) -nanodiscs. While the neutralizing epitopes in the purified Trx-gp41(662-723) were accessible and recognizable by anti-MPER bNAbs, these epitopes became less accessibly exposed, particularly in the C-terminal region of MPER, after incorporation of Trx-gp41(662-723) into nanodiscs. Highlights: HIV-1 gp41(662-723) was successfully expressed as a C-terminal fusion to the fusion partner Trx. Trx-gp41(662-723) was overexpressed into E. coli T7 Express lysY/I q by early induction and incubation at 20 °C/overnight. Trx-gp41(662-723) was successfully incorporated into a well-defined lipid nanodiscs. Negative-stained electron micrographs revealed homogenous Trx-gp41(662–723)-nanodiscs. The antigenicity of Trx-gp41(662-723) showed a dramatically decrease after incorporation into nanodiscs.