Purification Of Recombinant Single-chain Antibody BG4 And The Research Of Its Interaction With G-quadruplexes

G-quadruplexes are the three-dimensional DNA or RNA structures formed by guanine-tract rich sequences,with differential topological conformation.G-quadruplex motifs are generally present at the ends of chromosomes and in the promoter regions of many cancer-related genes,and possess regulatory activities.To date,characterized proteins with known ability to bind G-quadruplexes include zinc finger proteins and single chain antibody fragment(scFv).Each scFv is a protein with the variable region of an antibody's heavy chain and the variable region of its light chain linked by a peptide,which can be generated by the phage display technology.Currently,commercial scFv-type G-quadruplex antibodies,such as BG4,are available.Construction of expression vectors of G-quadruplex antibodies can produce large amounts of recombinant antibodies that can be used for various functional studies of G-quadruplexes.In this study,we constructed prokaryotic and eukaryotic expression vectors for the commercialized scFv-type G-quadruplex antibody BG4.The recombinant BG4 antibody was isolated and purified from a prokaryotic expression system.Ni-NTA beads were used to purify the BG4 antibody and an elution method with a gradient of imidazole concentrations was employed to remove non-specific bands and obtain the target protein with high purity.EMSA gel migration assay and immunofluorescence assay were used to verify the ability of the purified recombinant BG4 antibody to specifically bind G-quadruplexes in vitro.We also tested the experimental conditions the BG4 antibody in binding G-quadruplexes in vitro and its preservation conditions.The presence of the potassium ion could facilitate BG4 antibody's binding to G-quadruplex,while the reaction temperature and reaction time had limited effects on their binding.In addition,BG4 showed high stability and binding activity,and could be stored directly at-80?,In immunofluorescence experiments,the transfection of the MYC-G4 oligonucleotide and the presence of PDS(pyridostatin),a G-quadruplex stabilizer,resulted in increasing G-quadruplex formation in cells and strong detectable fluorescence signal,which further reinforced that the recombinant BG4 antibody could bind G-quadruplexes.Using lentiviral infection,the BG4 antibody and GFP-BG4 fusion protein could be stably expressed in cancer cells.No effects of the BG4 antibody expression on cell proliferation was observed,but it could alter the expression of different intracellular oncogenes,including downregulation of VEGFA expression and upregulation of MYC expression,but no significant effects on the expression of NRAS and HIF1? genes.Overall,we could produce large amounts of the recombinant BG4 antibody,which provided an important experimental basis for the visualization of G-quadruplexes.The expression of the BG4 antibody in eukaryotic cells and the characterization of its binding to intracellular G-quadruplexes are of great importance to the future functional studies of G-quadruplexes.