High-level expression of lacZ under control of the tac or trp promoter using runaway replication vectors in Escherichia coli.
To determine the utility of coupling runaway replication to the expression of cloned genes under the control of strong promoters, lacZ transcriptional fusions to the trp or tac promoter (Ptrp or Ptac) were constructed using plasmids in which the copy number is thermally regulated. Cells containing these plasmids were able to produce beta-galactosidase to levels between 3700 and 46,000 Miller units when induced only by a temperature upshift. The addition of the appropriate chemical inducer, either IPTG (isopropyl-beta-D-thiogalactopyranoside) or IAA (3-beta-indoleacrylic acid), did not significantly enhance the thermal induction. The Ptac-controlled and Ptrp-controlled lacZ induction differed slightly in that the Ptac-controlled thermal induction exhibited a lag of approximately 1.5 h as compared to both chemical and thermal induction, whereas in the case of Ptrp-controlled induction, an increase in beta-galactosidase expression above background occurred at approximately the same time regardless of the means of induction. The best vector, a Ptrp-controlled lacZ fusion carried on a runaway replication vector having a basal copy number of 10, was able to mediate the expression of beta-galactosidase to approximately 40,000 Miller units of beta-galactosidase comprising 25% of the total cell protein at 17 h postinduction under optimal conditions for protein yield. In these cells, lysis occurred as lacZ was maximally expressed. Under noninducing conditions, the plasmids were stable for at least 60 generations in the absence of antibiotic in batch culture.
Kidwell
J
J
Department of Biology, James Madison University, Harrisonburg, Virginia 22807, USA.
Kolibachuk
D
D
Dennis
D
D