Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: an application to overproduction of chicken lysozyme

A novel expression vector pKPl500 for synthesizing unfused proteinin Escherichia coli was constructed. pKP1500 perserves the tacpromoter, the lacZ SD sequence, unique restriction sites (EcoRI,SmaI, BamHI, SalI, PstI and HindIII) and the rrnB terminatorsof pKK223-3, but the replication origin is replaced with thatof pUC9. Construction of this plasmid is based upon the observationthat the copy number control of pUC9 is temperature dependent.At 28°C, the copy number of pKP1500 is less than 25 perchromosome, approximately the same copy number as that of pKK223-3,which contains the replication origin of pBR322, whereas at42°C, the copy number increases about 10 times and reachesup to 230 copies per chromosome. The main advantage of thissystem is that the temperature-dependent copy control and regulatableexpression of the tac promoter make cells car rying pKPl500derivatives stable against selective pressure by detrimentaloverproduction of foreign proteins at a low temperature andpermits high expression of cloned DNAs at a high temperature.When chicken lysozyme cDNA carrying the initiation codon (ATG)immediately upstream from the Lys¹ codon was inserted downstreamfrom the tac promoter and the SD sequence, the pKP1500 derivativeproduced lysozyme at about 25% of the total cellular proteins.This value is more than 10 times higher than that obtained withthe pKK223-3 derivative carrying the same lysozyine cDNA. Bycomparison, the expression of eukaryotic genes from the tacpromoter reported by others has usually been less than a few% of the total cellular protein. pKPl500 would therefore beuseful for the high level production of unfused proteins fromeukaryotic cDNAs in E. coli.