The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase.
Heterologous gene products produced by Escherichia coli cells can be exported into the culture medium by the action of the kil gene of the ColE1 plasmid, which encodes a bacterial release protein. The kil gene was fused with the stationary-phase promoter of the fic gene of E. coli, and a secretion cassette (Kil-Km cassette) containing the regulated kil gene, the Km-resistance gene, and multiple cloning sites for the integration of target genes was constructed. Using the gene for beta-glucanase (bgl) as a target gene, it was shown that the protein produced was only secreted into the medium during the stationary phase. Quasi-lysis and lethality were not observed. The primary effect of the induction of the kil gene was the overproduction of beta-glucanase. The total amount produced per milliliter of bacterial culture was almost threefold higher than that of the corresponding Kil- control. The protein pattern of periplasm and culture medium was analyzed before and after induction of the kil gene expression, indicating that the release of periplasmic proteins is semiselective. This secretion system is the first to use a growth-phase-regulated promoter for the expression of the kil gene.
Miksch
G
G
Abteilung Biotechnologie, Fakultät für Biowissenschaften, Pharmazie und Psychologie, Universität Leipzig, D-04318 Leipzig, Germany.
Fiedler
E
E
Dobrowolski
P
P
Friehs
K
K
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D054410:Chemokine CCL7; D003001:Cloning, Molecular; D016207:Cytokines / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005959:Glucosidases / Q000235:genetics / Q000378:metabolism; D017874:Immediate-Early Proteins / Q000235:genetics; D018945:Monocyte Chemoattractant Proteins; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D016196:S Phase / Q000235:genetics
D001426:Bacterial Proteins; D054410:Chemokine CCL7; D016207:Cytokines; D029968:Escherichia coli Proteins; D017874:Immediate-Early Proteins; D018945:Monocyte Chemoattractant Proteins; C069003:kil protein, E coli; D005959:Glucosidases; C066586:6-phospho-beta-glucosidase