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 pKP1500 for synthesizing unfused protein in Escherichia coli was constructed. pKP1500 perserves the tac promoter, the lacZ SD sequence, unique restriction sites (EcoRI, SmaI, BamHI, SalI, PstI and HindIII) and the rrnB terminators of pKK223-3, but the replication origin is replaced with that of pUC9. Construction of this plasmid is based upon the observation that the copy number control of pUC9 is temperature dependent. At 28 degrees C, the copy number of pKP1500 is less than 25 per chromosome, approximately the same copy number as that of pKK223-3, which contains the replication origin of pBR322, whereas at 42 degrees C, the copy number increases about 10 times and reaches up to 230 copies per chromosome. The main advantage of this system is that the temperature-dependent copy control and regulatable expression of the tac promoter make cells carrying pKP1500 derivatives stable against selective pressure by detrimental overproduction of foreign proteins at a low temperature and permits 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 downstream from the tac promoter and the SD sequence, the pKP1500 derivative produced lysozyme at about 25% of the total cellular proteins. This value is more than 10 times higher than that obtained with the pKK223-3 derivative carrying the same lysozyme cDNA. By comparison, the expression of eukaryotic genes from the tac promoter reported by others has usually been less than a few % of the total cellular protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Miki
T
T
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Yasukochi
T
T
Nagatani
H
H
Furuno
M
M
Orita
T
T
Yamada
H
H
Imoto
T
T
Horiuchi
T
T
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003470:Culture Media; D004247:DNA / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005822:Genetic Vectors; D009113:Muramidase / Q000235:genetics*; D009154:Mutation; D009841:Oligonucleotides / Q000138:chemical synthesis; D010957:Plasmids
D003470:Culture Media; D009841:Oligonucleotides; D004247:DNA; D009113:Muramidase