title: Optimal production of a fusion protein consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in fed-batch culture of Pichia pastoris.
abstract: OBJECTIVE
  The aim of the present study was to establish the strategy for producing a single-chain variable fragment (scFv) antibody fused with interleulin-2 (IL2) by Pichia pastoris and to optimize production during fed-batch cultivation in a 5-l fermenter.
  
  
  METHODS
  We constructed a fusion sequence consisting of an scFv gene derived from a mouse monoclonal antibody against a tumor-associated antigen (designated MK-1 antigen) and human interleulin-2 (IL-2) gene, ligated the sequences to expression vector pPICZα-A and separately transformed the constructs into Pichia pastoris strains GS115 and KM71H.
  
  
  RESULTS
  The highest concentration of secreted fusion protein, 738 ± 44 mg/l, was obtained after a 60-h induction. To investigate the specific binding activity of the partially purified fusion protein, we used an enzyme-linked immunosorbent assay and antigen from a whole-cell lysate. Student's t-test showed that the specific binding activity of the partially-purified fusion protein to the lysate of Chinese hamster ovary cell lines expressing the MK-1 antigen was significantly higher than that of the lysate of CHO cell lines that do not express MK-1.
  
  
  CONCLUSIONS
  The method described here permits the production of substantial amounts of the fusion protein for conducting functional studies on the biological role of these fusion proteins.
authors: Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
authors: Sakorn
authors: S
authors: Anuleejun
authors: Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
authors: Tanapat
authors: T
authors: Palaga
authors: Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Japan.
authors: Yoshio
authors: Y
authors: Katakura
authors: Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Japan School of Nursing, Faculty of Medicine, Fukuoka University, Fukuoka, Japan Department of Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
authors: Motomu
authors: M
authors: Kuroki
authors: School of Nursing, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
authors: Masahide
authors: M
authors: Kuroki
authors: Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand suchada.cha@chula.ac.th.
authors: Suchada Chanprateep
authors: SC
authors: Napathorn
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology*; D061249:Batch Cell Culture Techniques; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000276:immunology*; D002846:Chromatography, Affinity; D003412:Cricetulus; D000071858:Epithelial Cell Adhesion Molecule; D006863:Hydrogen-Ion Concentration; D007376:Interleukin-2 / Q000276:immunology*; D010843:Pichia / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000096:biosynthesis*; D013696:Temperature
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies
keywords: Fusion protein; IL2; single-chain variable fragment; tumor-associated antigen