Expression, purification, and isotope labeling of the Fv of the human HIV-1 neutralizing antibody 447-52D for NMR studies

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 pe1B 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 pe1B 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.