In silico design and in vitro characterization of a recombinant antigen for specific recognition of NMP22

Abstract

Although urine cytology and cystoscopy are current gold standard methods in diagnosis and surveillance of Bladder cancer (BC), they have some limitations which necessitates novel diagnostic approaches to compensate their drawbacks. In this regard, Nuclear Matrix Protein 22 (NMP22) is introduced as a potential tumor biomarker for BC detection (FDA approved). NMP22 determination mainly occurs through immunoassay platforms, raising a proper antibody against its antigen. Hence, development of such immunoassays seems crucial. Various bioinformatic tools were harnessed to select a region with lowest variability, highest density for linear and conformational epitopes, lowest post translational modifications, highest antigenicity, best physicochemical properties and reliable transcriptional properties. Subsequently, E. coli BL21 (DE3) and P. pastoris GS115 were applied for exogenous expression. Ultimately, protein purification and quantification was followed by ELISA test for antibody analyses. Both host successfully expressed the antigen, while the E. coli expression was with higher yield. The commercial anti-NMP22 antibodies showed relatively equal detection results. However, the slight better detection for the antigen with P. pastoris origin could be deduced as better structural properties for P. pastoris. These results indicate higher expression yields and lower costs for over-expression of this eukaryotic antigen.

Keywords: Antibody; Bioinformatics; Immunoassay; NMP22; Protein expression.