optimized expression and purification of humbug in pichia pas¬toris and its monoclonal antibody preparation

background: the humbug gene is a truncated isoform of aspartyl β-hydroxylase (asph) gene that is overexpressed in many human malignancies. in recent years, since humbug has received increasing attention, it is considered as a potential therapeutic molecular target. therefore, it is necessary for preparing humbug protein and its monoclonal antibody to investigate its structure and function. method: the optimized humbug gene, synthesized by genscript in nanjing, china on december 21st 2013, was expressed in pichia pastoris cells that were cultured in a 10-l bioreactor. the recombinant protein was further obtained and purified by using ion exchange chromatography and sephadex g75. the humbug protein was used to immunize balb/c mice to generate the monoclonal antibodies. the specificity and sensitivity of the monoclonal antibodies were assessed by indirect enzyme-linked immunosorbent assay. finally, the humbug monoclonal antibodies were used to detect the expression of humbug in several tumor cell lines via indirect immunofluorescence. results: firstly, the recombinant humbug was expressed in p. pastoris successfully and efficiently by using a gene-optimized strategy. secondly, the purification process of humbug was established via multiple chromatography methods. in addition, four monoclonal antibodies against humbug were obtained from the immunized balb/c mice, and the result of indirect immunofluorescence was indicated that the humbug monoclonal antibody showed the high affinity with humbug protein, which expressed in several tumor cell lines. conclusion: the over-expression of recombinant humbug provides adequate sources for its structural study and the preparation of the humbug-specific monoclonal antibody can potentially be used in tumor initial diagnosis and immunotherapy.