title: The order of expression is a key factor in the production of active transglutaminase in Escherichia coli by co-expression with its pro-peptide.
abstract: BACKGROUND
  Streptomyces transglutaminase (TGase) is naturally synthesized as zymogen (pro-TGase), which is then processed to produce active enzyme by the removal of its N-terminal pro-peptide. This pro-peptide is found to be essential for overexpression of soluble TGase in E. coli. However, expression of pro-TGase by E. coli requires protease-mediated activation in vitro. In this study, we developed a novel co- expression method for the direct production of active TGase in E. coli.
  
  
  RESULTS
  A TGase from S. hygroscopicus was expressed in E. coli only after fusing with the pelB signal peptide, but fusion with the signal peptide induced insoluble enzyme. Therefore, alternative protocol was designed by co-expressing the TGase and its pro-peptide as independent polypeptides under a single T7 promoter using vector pET-22b(+). Although the pro-peptide was co-expressed, the TGase fused without the signal peptide was undetectable in both soluble and insoluble fractions of the recombinant cells. Similarly, when both genes were expressed in the order of the TGase and the pro-peptide, the solubility of TGase fused with the signal peptide was not improved by the co-expression with its pro-peptide. Interestingly, active TGase was only produced by the cells in which the pro-peptide and the TGase were fused with the signal peptide and sequentially expressed. The purified recombinant and native TGase shared the similar catalytic properties.
  
  
  CONCLUSIONS
  Our results indicated that the pro-peptide can assist correct folding of the TGase inter-molecularly in E. coli, and expression of pro-peptide prior to that of TGase was essential for the production of active TGase. The co-expression strategy based on optimizing the order of gene expression could be useful for the expression of other functional proteins that are synthesized as a precursor.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Lihu Avenue, Wuxi, China.
authors.forename: Song
authors.initials: S
authors.lastname: Liu
authors.forename: Dongxu
authors.initials: D
authors.lastname: Zhang
authors.forename: Miao
authors.initials: M
authors.lastname: Wang
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cui
authors.forename: Kangkang
authors.initials: K
authors.lastname: Chen
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D011503:Transglutaminases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011503:Transglutaminases