title: Expression, purification, and isotope labeling of the Fv of the human HIV-1 neutralizing antibody 447-52D for NMR studies.
abstract: The Fv is the smallest antigen binding fragment of the antibody and is made of the variable domains of the light and heavy chains, V(L) and V(H), respectively. The 26-kDa Fv is amenable for structure determination in solution using multi-dimensional hetero-nuclear NMR spectroscopy. The human monoclonal antibody 447-52D neutralizes a broad spectrum of HIV-1 isolates. This anti-HIV-1 antibody elicited in an infected patient is directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. The V3 loop is an immunodominant neutralizing epitope of HIV-1. To obtain the 447-52D Fv for NMR studies, an Escherichia coli bicistronic expression vector for the heterodimeric 447-52D Fv and vectors for single chain Fv and individually expressed V(H) and V(L) were constructed. A pelB signal peptide was linked to the antibody genes to enable secretion of the expressed polypeptides into the periplasm. For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pelB sequence and following the termination codon. A set of oligonucleotides that prime the leader peptide genes of all potential antibody human antibodies were designed as backward primers. The forward primers for the V(L) and V(H) were based on constant region sequences. The 447-52D Fv could not be expressed either by a bicistronic vector or as single chain Fv, probably due to its toxicity to Escherichia coli. High level of expression was obtained by individual expression of the V(H) and the V(L) chains, which were then purified and recombined to generate a soluble and active 447-52D Fv fragment. The V(L) of mAb 447-52D was uniformly labeled with 13C and 15N nuclei (U-13C/15N). Preliminary NMR spectra demonstrate that structure determination of the recombinant 447-52D Fv and its complex with V3 peptides is feasible.
authors.affiliation: Department of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Naama
authors.initials: N
authors.lastname: Kessler
authors.forename: Anat
authors.initials: A
authors.lastname: Zvi
authors.forename: Min
authors.initials: M
authors.lastname: Ji
authors.forename: Michal
authors.initials: M
authors.lastname: Sharon
authors.forename: Osnat
authors.initials: O
authors.lastname: Rosen
authors.forename: Rina
authors.initials: R
authors.lastname: Levy
authors.forename: Miroslaw
authors.initials: M
authors.lastname: Gorny
authors.forename: Suzan
authors.initials: S
authors.lastname: Zolla-Pazner
authors.forename: Jacob
authors.initials: J
authors.lastname: Anglister
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D015483:HIV Antibodies / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D002247:Carbon Isotopes; D015483:HIV Antibodies; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins