title: Optimized Expression and Purification of Humbug in Pichia pastoris and Its Monoclonal Antibody Preparation.
abstract: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Key Laboratory for Space Bioscience and Space Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Huyan
authors.affiliation: Key Laboratory for Space Bioscience and Space Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.
authors.forename: Ruihua
authors.initials: R
authors.lastname: Tang
authors.affiliation: Key Laboratory for Space Bioscience and Space Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: Key Laboratory for Space Bioscience and Space Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.affiliation: Key Laboratory for Space Bioscience and Space Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Xue
authors.affiliation: Key Laboratory for Space Bioscience and Space Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
keywords: Fermentation; Humbug; Monoclonal antibody; Pichia pastoris; Tumor diagnosis
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