title: An efficient production and characterization of HIV-1 gp41 ectodomain with fusion peptide in Escherichia coli system.
abstract: We demonstrated a high level expression and purification of recombinant human immunodeficiency virus type 1 gp41 ectodomain (gp41e-FP) using glass bead approach with a final yield of 12±2mg/L bacterial culture. The proper folding of gp41e-FP encompassing the fusion peptide (FP) was ascertained by circular dichroism (CD) measurement and recognition by NC-1 antibody. The latter assay revealed stabilization of the gp41 coiled coil structure in the presence of liposome dispersion. The differential affinity of gp41e-FP and gp41e (devoid of FP) by NC-1 suggested an aggregated state for gp41e-FP and/or possible proximity of the fusion peptide domain to the coiled coil structure of gp41 ectodomain. Perfluorooctanoate (PFO)-PAGE electrophoresis experiment revealed the trimeric propensity of the recombinant gp41e-FP. In comparison to gp41e, the lipid mixing activity of gp41e-FP was two-fold higher suggesting a role of FP in promoting membrane fusion. The present approach to efficiently and quantitatively preparing the functional full-length recombinant gp41 ectodomain protein can be employed for structural and biomedical investigations and the extraction of other inclusion body-embedded recombinant proteins.
authors.affiliation: Institute of Chemistry, Academia Sinica, 128, Academia Road, Sec 2, Nankang, Taipei 11529, Taiwan, ROC.
authors.forename: Chun-Hung
authors.initials: CH
authors.lastname: Lin
authors.forename: Chi-Hui
authors.initials: CH
authors.lastname: Lin
authors.forename: Chung-Chieh
authors.initials: CC
authors.lastname: Chang
authors.forename: Ting-Shyang
authors.initials: TS
authors.lastname: Wei
authors.forename: Shu-Fang
authors.initials: SF
authors.lastname: Cheng
authors.forename: Steve S-L
authors.initials: SS
authors.lastname: Chen
authors.forename: Ding-Kwo
authors.initials: DK
authors.lastname: Chang
mesh_terms: D001709:Biotechnology / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D020349:Surface Plasmon Resonance
chemical_list: D015700:HIV Envelope Protein gp41; D008563:Membrane Lipids; D010455:Peptides; D011993:Recombinant Fusion Proteins