title: Evaluation of bottlenecks in proinsulin secretion by Escherichia coli.
abstract: This work evaluates three potential bottlenecks in recombinant human proinsulin secretion by Escherichia coli: protein stability, secretion capacity and the effect of molecular size on secretion efficiency. A maximum secretion level of 7.2 mg g(-1) dry cell weight was obtained in the periplasm of E. coli JM109(DE3) host cells. This value probably represents an upper limit in the transport capacity of E. coli cells secreting ZZ-proinsulin and similar proteins with the protein A signal peptide. A selective deletion study was performed in the fusion partner and no effect of the molecular size (17-24 kDa) was detected on secretion efficiency. The protective effect against proteolysis provided by the ZZ domain was thoroughly demonstrated in the periplasm of E. coli and it was also shown that a single Z domain is able to provide the same protection level without compromising the downstream processing. The use of this shorter fusion partner enables a 1.6-fold increase in the recovery of the target protein after cleavage of the affinity handle.
authors.affiliation: Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
authors.forename: F J M
authors.initials: FJ
authors.lastname: Mergulhão
authors.forename: M A
authors.initials: MA
authors.lastname: Taipa
authors.forename: J M S
authors.initials: JM
authors.lastname: Cabral
authors.forename: G A
authors.initials: GA
authors.lastname: Monteiro
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019897:Periplasm / Q000378:metabolism; D011384:Proinsulin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D011384:Proinsulin