High-level expression of a semisynthetic dam gene in Escherichia coli.
We constructed a semisynthetic gene encoding a DNA-adenine-methyltransferase (Dam) that codes for the same amino acid sequence as the wild type (wt) Escherichia coli dam gene. Since for unknown reasons the entire wt sequence, from the start codon to the end of the gene, could not be cloned, a gene was constructed consisting of a chemically synthesized 5' portion and a 3' portion from the E. coli chromosome. Introduction of this semisynthetic gene into a suitable vector allows overproduction of E. coli Dam in mg amounts per liter E. coli culture, with optimum expression of the gene in the vector pJLA503. This plasmid places the target gene under control of the strong, tandemly arranged pR pL promoters from bacteriophage lambda, regulated by a temperature-sensitive lambda repressor. A rapid, two-column purification protocol is described that allows for very fast purification of the protein. The 32-kDa recombinant protein methylates the sequence GATC.
Hülsmann
K H
KH
Institut für Kristallographie, Freie Universität Berlin, F.R.G.
Quaas
R
R
Georgalis
Y
Y
Saenger
W
W
Hahn
U
U
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)