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 expres sion of cloned genes under the control of strong promoters, lacZ trans criptional fusions to the trp or tac promoter (Ptrp or Ptac) were cons tructed using plasmids in which the copy number is thermally regulated . Cells containing these plasmids were able to produce beta-galactosid ase to levels between 3700 and 46,000 Miller units when induced only b y a temperature upshift. The addition of the appropriate chemical indu cer, either IPTG (isopropyl-beta-o-thiogalactopyranoside) or IAA (3-be ta-indoleacrylic acid), did not significantly enhance the thermal indu ction. The Ptac-controlled and Ptrp-controlled lacZ induction differed slightly in that the Ptac-controlled thermal induction exhibited a la g of approximately 1.5 h as compared to both chemical and thermal indu ction, whereas in the case of Ptrp-controlled induction, an increase i n beta-galactosidase expression above background occurred at approxima tely the same time regardless of the means of induction. The best vect or, a Ptrp-controlled lacZ fusion carried on a runaway replication vec tor having a basal copy number of 10, was able to mediate the expressi on of beta-galactosidase to approximately 40,000 Miller units of beta- galactosidase comprising 25% of the total cell protein at 17 h postind uction under optimal conditions for protein yield. In these cells, lys is occurred as lacZ was maximally expressed. Under noninducing conditi ons, the plasmids were stable for at least 60 generations in the absen ce of antibiotic in batch culture. (C) 1996 John Wiley & Sons, Inc.