title: Functional expression of recombinant human trefoil factor 1 by Escherichia coli and Brevibacillus choshinensis.
abstract: BACKGROUND
  Trefoil factor 1 (TFF1) mediates mucosal repair and belongs to a highly conserved trefoil factor family proteins which are secreted by epithelial cells in the stomach or colon mucous membrane. TFF1 forms a homodimer via a disulphide linkage that affects wound healing activity. Previous recombinant expressions of TFF1 were too low yield for industrial application. This study aims to improve the expression level of bioactive recombinant TFF1 (rTFF1) and facilitate application potency.
  
  
  METHODS
  The rTFF1 gene rtff1 was synthesized, expressed by Escherichia coli and secreted by Brevibacillus choshinensis. The rTFF1s were purified. The polymeric patterns and wound healing capacities of purified rTFF1s were checked.
  
  
  RESULTS
  In Escherichia coli, 21.08 mg/L rTFF1 was stably expressed as monomer, dimer and oligomer in soluble fraction. In Brevebacillus choshinensis, the rTFF1 was secreted extracellularly at high level (35.73 mg/L) and formed monomer, dimer and oligomer forms. Both proteins from different sources were purified by Ni-NTA chromatography and exhibited the wound healing activities. The rTFF1 produced by B. choshinensis had better wound healing capability than the rTFF1 produced by E. coli. After pH 2.4 buffer treatments, the purified rTFF1 formed more oligomeric forms as well as better wound healing capability. Glycosylation assay and LC-MS/MS spectrometry experiments showed that the rTFF1 produced by B. choshinensis was unexpectedly glycosylated at N-terminal Ser residue. The glycosylation may contribute to the better wound healing capacity.
  
  
  CONCLUSIONS
  This study provides a potent tool of rTFF1 production to be applied in gastric damage protection and wound healing. The protein sources from B. choshinensis were more efficient than rTFF1 produced by E. coli.
authors.affiliation: Department of Food Science and Biotechnology, National Chung-Hsing University, Taichung, Taiwan, Republic of China. q4351@yahoo.com.tw.
authors.forename: Yueh-Mei
authors.initials: YM
authors.lastname: Cheng
authors.affiliation: Department of Food Science and Biotechnology, National Chung-Hsing University, Taichung, Taiwan, Republic of China. sandy19870710@hotmail.com.
authors.forename: Meng-Ting
authors.initials: MT
authors.lastname: Lu
authors.affiliation: Department of Food Science and Biotechnology, National Chung-Hsing University, Taichung, Taiwan, Republic of China. cmyeh@nchu.edu.tw.|Agricultural Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan, Republic of China. cmyeh@nchu.edu.tw.
authors.forename: Chuan Mei
authors.initials: CM
authors.lastname: Yeh
mesh_terms: D058675:Brevibacillus / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005753:Gastric Mucosa / Q000187:drug effects / Q000293:injuries; D015870:Gene Expression; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D014945:Wound Healing / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; C051838:TFF1 protein, human; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins