Sensing prothymosin alpha origin, mutations and conformation with monoclonal antibodies

• Published in: Journal of immunological methods 2002-08, Vol.266 (1), p.185-196
• Main Authors: Sukhacheva, Elena A, Evstafieva, Alexandra G, Fateeva, Tatyana V, Shakulov, Vitaliy R, Efimova, Nadezda A, Karapetian, Ruben N, Rubtsov, Yuri P, Vartapetian, Andrey B
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - immunology; Antibody Specificity; Epitope Mapping; Gene Products, tat - genetics; Green fluorescent protein; Green Fluorescent Proteins; HeLa Cells; Humans; Luminescent Proteins - genetics; Mice; Mice, Inbred BALB C; Monoclonal antibody; Point Mutation; Protein Conformation; Protein Precursors - chemistry; Protein Precursors - genetics; Protein Precursors - immunology; Prothymosin α; Recombinant Fusion Proteins - immunology; Species Specificity; Tat transduction peptide; Thymosin - analogs & derivatives; Thymosin - chemistry; Thymosin - genetics; Thymosin - immunology
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(02)00098-4

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.