title: High expression of fusion proteins consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in Escherichia coli.
abstract: OBJECTIVE
  The aim of this study was to establish a strategy for high-level production of single-chain variable fragment (scFv) antibodies fused with interleukin-2 (IL-2) in Escherichia coli.
  
  
  METHODS
  We constructed two fusion sequences consisting of a scFv gene derived from a mouse monoclonal antibody against a tumor-associated antigen (MK-1) and human Interleukin-2(IL-2) gene, ligated the fusions into pET15b and transformed into three different E. coli strains. The effects of temperature, isopropyl-β-D-thiogalactopyranoside (IPTG) concentration and duration of IPTG induction were investigated.
  
  
  RESULTS
  Employing E. coli strain Rosetta-gami B, which has an oxidizing cytoplasm that facilitates cytoplasmic disulfide bond formation, improved the level of soluble protein expression. Under optimal conditions, the highest levels of fusion protein expression and high percentages of the proteins were found in their soluble form. Specifically, 89.29% (0.28 g/l) of one fusion protein was soluble after a 10-h induction and 84.61% (0.26 g/l) of the other fusion protein was soluble after a separate 10-h induction. When analyzed by enzyme-linked immunosorbent assay, the partially-purified fusion proteins retained a specific binding activity to the cell lysate of Chinese hamster ovary (CHO) cells expressing MK-1.
  
  
  CONCLUSIONS
  Taken together, the methods described herein permit the production of substantial amounts of the fusion proteins for conducting functional studies on the biological role of these fusion proteins.
authors: Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand suchada.cha@chula.ac.th.
authors: Suchada Chanprateep
authors: SC
authors: Napathorn
authors: School of Nursing, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
authors: Motomu
authors: M
authors: Kuroki
authors: Department of Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
authors: Masahide
authors: M
authors: Kuroki
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology*; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000276:immunology*; D003412:Cricetulus; D000071858:Epithelial Cell Adhesion Molecule; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology*; D010957:Plasmids; 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; IL-2; MK-1; single-chain variable fragment; tumor-associated antigen