title: Expression of metallocarboxypeptidase inhibitors in Escherichia coli: effect of cysteine content and protein size in the secretory production of disulfide-bridged proteins.
abstract: Metallocarboxypeptidase inhibitors are proteins with possible applications in biomedicine given their properties as anticoagulant and antitumoral factors. They are small, eukaryotic polypeptides comprising several disulfide bridges, which makes them hard to express in inexpensive bacterial hosts. In this work, three of them were produced in high-cell-density cultures of Escherichia coli: PCI (39 residues and three bridges), LCI (66 residues and four bridges) and TCI (75 residues and six bridges). The genes coding for the mentioned inhibitors were cloned in an arabinose-inducible plasmid fused to the signal peptide of DsbA in order to have them secreted and grant the formation of the bridges. The trigger-factor defective strain KTD101 was used as the expression host. The resulting recombinant strains were cultured in fed-batch mode employing minimal media and an exponential feed profile, keeping the specific growth rate at μ = 0.1 h(-1) by limitation of the fed carbon source (glycerol). Between 380 and 540 mg l(-1) of active inhibitors were obtained in both the periplasmic extracts and extracellular media of the cultures. Later on, excretion was enhanced using a cell permeabilization treatment, allowing the recovery of over 80% of the products from the extracellular fraction. Protein yields were found to be inversely proportional to cysteine content of the inhibitor, whereas protein excretion rates were inversely proportional to the protein size. Overall, these results offer insight into the secretory production of active disulfide-bridged proteins in high-cell-density cultures of E. coli.
authors.affiliation: Unitat de Biocatàlisi Aplicada associada al IQAC, Departament d'Enginyeria Química, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Edifici Q, 08193, Bellaterra, Spain. juanmiguel.puertas@uab.cat
authors.forename: Juan-Miguel
authors.initials: JM
authors.lastname: Puertas
authors.forename: Glòria
authors.initials: G
authors.lastname: Caminal
authors.forename: Glòria
authors.initials: G
authors.lastname: González
mesh_terms: D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D003545:Cysteine / Q000032:analysis / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D055571:Secretory Pathway
chemical_list: D004220:Disulfides; C116332:LCI protein, Hirudo medicinalis; D011480:Protease Inhibitors; D021382:Protein Sorting Signals; D011506:Proteins; D011994:Recombinant Proteins; D002268:Carboxypeptidases; D003545:Cysteine