Sensing prothymosin alpha origin, mutations and conformation with monoclonal antibodies.

Abstract To overcome poor immunogenicity of prothymosin α, a small and highly acidic nuclear protein involved in cell proliferation, production of anti-prothymosin α antibodies in mice immunized with free human prothymosin α, with prothymosin α coupled to different carriers and with prothymosin α fused to green fluorescent protein was assessed. Fusing prothymosin α to green fluorescent protein turned out to be the superior approach resulting in production of high titer anti-prothymosin α antibodies. From these studies, two highly specific anti-prothymosin α monoclonal antibodies recognizing epitopes within the amino terminal (2F11) and middle (4F4) portions of the human prothymosin α molecule were obtained and characterized. As expected, the 2F11 antibody displayed broad species specificity, whereas the 4F4 antibody appeared to be species-specific permitting discrimination of human versus rat protein. Furthermore, a combination of point mutations in prothymosin α that alter the properties of the protein precluded recognition by the 4F4 antibody. Intramolecular masking of the 4F4 epitope in prothymosin α fused to the Tat transduction peptide of human immunodeficiency virus type 1 was observed. The anti-prothymosin α antibodies obtained were suitable for precipitation of human prothymosin α from HeLa cell lysates and for immunolocalization of the endogenous prothymosin α within the cells. Fusion with green fluorescent protein may thus be helpful in raising antibodies against ‘problematic’ proteins.