Controlled specific release of periplasmic proteins from genetically engineered Escherichia coli strains

The kil gene of pMB9 under the control of the tightly regulated leftward promoter (pL) of coliphage lambda has been cloned. Three types of plasmids were constructed. In all cases the activity of the lambda promoter is controlled by a thermosensitive cI repressor (product of the cI857 gene) supplied from a resident defective prophage or cloned onto a compatible p15A derived plasmid. Induction of the Kil protein is brought about by a temperature shift of the culture from 28 degrees Celsius to 42 degrees Celsius. Plasmid pPLc28K1 contains the kil gene situated behind its natural ribosome binding site and preceded by a transcription termination site. Using a bacterial strain with anti-termination properties (e.g. M5219) periplasmic proteins can upon induction be gradually released into the medium without severe inhibition of the growth of the host strain. The second plasmid, pPLc321, contains the kil coding sequence preceeded by an engineered ribosome binding site derived from the Escherichia coli tryptophan operon attenuator. With this plasmid induction of the Kil protein is very rapid and specific release of the periplasmic proteins is essentially complete within 30 min after induction. In a third construct, pcI857K1, the pL - kil casette together with the cI857 allele are present on the same replicon, a derivative of p15A, 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 temperature raise 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.