The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secre
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© Springer-Verlag 1997
O R I G I N A L PA P E R
G. Miksch · E. Fiedler · P. Dobrowolski · K. Friehs
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
Received: 17 October 1996 / Accepted: 13 December 1996
Abstract 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 β-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 β-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. Key words Protein secretion · kil Gene · β-Glucanase · Escherichia coli · Growth-phase-dependent promoter · Stationary phase
G. Miksch (Y) · E. Fiedler · P. Dobrowolski1 Abteilung Biotechnologie, Fakultät für Biowissenschaften, Pharmazie und Psychologie, Universität Leipzig, D-04318 Leipzig, Germany Tel. +49-341-2352078; Fax +49-341-2352078 K. Friehs AG Fermentationstechnik, Technische Fakultät, Universität Bielefeld, D-33615 Bielefeld, Germany 1 Kriminaltechnisches
Institut, LKA Sachsen, D-01111 Dresden, Germany
Introduction The secretion of proteins of interest into the culture medium offers numerous advantages for several reasons. It has been shown that proteolysis of secreted proteins is limited (Stader and Silhavy 1990; Kresze 1991). Furthermore, in gram-negative bacteria, the purification of proteins from the medium is relatively easy since the medium contains fewer proteins than lysed cells (Pugsley 1989; Stader and Silhavy 1990). The formation of intracellular protein aggregates or inclusion bodies upon high-level expression can be prevented by the secretion of proteins (Schoner et al. 1985; Lloubes et al. 1993). In addition, disulfide bridges are formed more easily when heterologous proteins are released into the culture medium (Hsiung et al. 1986; Kitai et al. 1988; Takagi et al. 1988; Plückthun 1991; Molina et al. 1992). Escherichia coli secretes only enterotoxins, fimbrial subunits, hemolysin, and bacteriocins naturally into the culture medium (Nicaud et al. 1986; Pugsley 1993)
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