Efficient specific release of periplasmic proteins from Escherichia coli using temperature induction of cloned kil gene of pMB9.
We have cloned the kil gene of pMB9 under control of the tightly regulated leftward promoter (pL) of coliphage lambda. Three types of plasmids were constructed. In all cases the activity of the lambda promoter is controlled by a thermosensitive cl repressor (product of the c/857 gene) supplied form a resident defective prophage or cloned onto a compatible p 15A-derived plasmid. Induction of the kil protein is brought about by a temperature shift of the culture from 28 degrees C to 42 degrees C. Plasmid pPLc28K1 contains the kil gene including its natural ribosome-binding site and preceded by a transcription termination site. Using a bacterial strain with antitermination properties (e.g., M5219), periplasmic proteins can upon induction be gradually the growth of the host strain. The second plasmid pPLc321K1, contains the kil-coding sequence preceded by an engineered ribosome binding site derived from the attenuator of the Escherichia coli tryptophan operon. With this plasmid induction of the Kil protein is very rapid and specific release of the periplasmic proteins in essentially complete within 30 min after induction. In a third construct, pcl857K1, the pL-kil cassette together with c/857 allele are present on the same replicon, which is compatible with ColE1-derived expression vectors. This configuration allows accumulation in the periplasm of cloned gene products, induced by, e.g., tac or trp promoters at low temperature and subsequent release into the medium following increase of the temperature of the culture. Under repressed conditions (growth at low temperature) all plasmids are perfectly stable in a large number of E. coli strains tested, also when cultivated on a 20-L fermentor scale. Controlled, heat-induced release of periplasmic proteins is highly specific and applicable at relatively high cell densities. The method therefore is an attractive alternative to cumbersome osmotic shock procedures for large-scale cultures.
Steidler
L
L
Laboratory of Molecular Biology, University of Gent, KL Ledeganckstraat 35, B-900 Gent, Belgium.
Fiers
W
W
Remaut
E
E