Isolation of gram quantities of EcoRI restriction and modification enzymes from an overproducing strain.
Structural genes for EcoRI restriction endonuclease and modification methylase have been inserted into the plasmid vector pKC30 (Shimatake, H., and Rosenberg, M. (1981) Nature (Lond.) 292, 128-132) downstream from the bacteriophage lambda pL promoter. Upon induction of pL expression in strains producing a thermolabile lambda cI857 repressor, synthesis of EcoRI polypeptides is enhanced to the extent that after 4 h they represent several per cent of the total cell protein. Purification of activities overproduced in this manner yields preparations of endonuclease and methylase which appear identical to those obtained from conventional sources, with overall yields corresponding to 0.5 to 0.9 g of each enzyme/kg of cell paste.
D004262:DNA Restriction Enzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D004790:Enzyme Induction; D004926:Escherichia coli; D005796:Genes; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
D004274:DNA, Recombinant; C020098:DNA modification methylase EcoRI; D008780:Methyltransferases; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI