title: A novel Ecotin-Ubiquitin-Tag (ECUT) for efficient, soluble peptide production in the periplasm of Escherichia coli.
abstract: BACKGROUND
  Many protocols for recombinant production of peptides and proteins include secretion into the periplasmic space of Escherichia coli, as they may not properly fold in the cytoplasm. If a signal peptide is not sufficient for translocation, a larger secretion moiety can instead be fused to the gene of interest. However, due to the covalent linkage of the proteins, a protease recognition site needs to be introduced in between, altering the N-terminus of the product. In the current study, we combined the ubiquitin fusion technology, which allows production of authentic peptides and proteins, with secretion by the perpiplasmic protease inhibitor ecotin.
  
  
  RESULTS
  Different fusion constructs, composed of ecotin, mouse ubiquitin b and a model peptide, were expressed in E. coli BL21(DE3). The fusion proteins were translocated into the periplasmic space and the ecotin signal peptide was cleaved off. Under the control of the lacUV5 promoter at 24 degrees C we obtained 18 mg periplasmic recombinant protein per gram dry cell weight. However, vigorous expression with the T7 promoter caused outer membrane permeabilization and leakage of the fusion protein into the culture medium. Target peptides were released from hybrid proteins by the deubiquitinating enzyme ubiquitin c-terminal hydrolase-L3 in vitro. MALDI TOF-TOF mass spectroscopy confirmed accurate cleavage.
  
  
  CONCLUSIONS
  This newly described method represents a useful technique for the production of authentic soluble peptides in the periplasm of E. coli. In addition, larger proteins might also be produced with the current system by the use of ubiquitin specific proteases, which can cleave off larger C-terminal extensions.
authors.affiliation: Austrian Center of Biopharmaceutical Technology, Muthgasse 18, A-1190 Vienna, Austria. Bernhard.Auer@uibk.ac.at.
authors.forename: Michael
authors.initials: M
authors.lastname: Paal
authors.forename: Thomas
authors.initials: T
authors.lastname: Heel
authors.forename: Rainer
authors.initials: R
authors.lastname: Schneider
authors.forename: Bernhard
authors.initials: B
authors.lastname: Auer