title: Expression, purification, and isotope labeling of a gp120 V3 peptide and production of a Fab from a HIV-1 neutralizing antibody for NMR studies.
abstract: Most human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies in infected individuals and in immunized animals are directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. This loop plays a crucial role in phenotypic determination, cytopathicity (syncytium induction), and coreceptor usage of HIV-1. The human monoclonal antibody 447-52D was found to neutralize a broad spectrum of HIV-1 strains. In order to solve the solution structure of the V3MN peptide bound to the 447-52D Fab fragment by NMR, large quantities of labeled peptide and a protocol for the purification of the Fab fragment were needed. An expression plasmid coding for the 23-residue V3 peptide of the HIV-1MN strain (V3MN peptide, YNKRKRIHIGPGRAFYTTKNIIG) linked to a derivative of the RNA-binding domain of hnRNCP1 was constructed. The fusion protein attached to the V3 peptide prevents its degradation. Using this system, U-15N, U-13C,15N, and U-13C,15N, 50% 2H labeled fusion protein molecules were expressed in Escherichia coli grown on rich Celtone medium with yields of about 240 mg/liter. The V3MN peptide was released by CNBr cleavage and purified by RP-HPLC, giving final yields of 6-13 mg/liter. This expression system is generally applicable for biosynthesis of V3-related peptides and was also used to prepare the V3JR-FL. The 447-52D Fab fragment was obtained by a short enzymatic papain cleavage of the whole antibody. Preliminary NMR spectra demonstrate that full structural analysis of the V3MN complexed to the 447-52D Fab is feasible. This system enables studies of the same epitope bound to different HIV-1 neutralizing antibodies.
authors.affiliation: Department of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Sharon
authors.forename: Matthias
authors.initials: M
authors.lastname: Görlach
authors.forename: Rina
authors.initials: R
authors.lastname: Levy
authors.forename: Yehezkiel
authors.initials: Y
authors.lastname: Hayek
authors.forename: Jacob
authors.initials: J
authors.lastname: Anglister
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D015483:HIV Antibodies / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000276:immunology; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments