Isolation of a strain overproducing endonuclease Eco29kI: purification and characterization of the homogeneous enzyme.
The physical map of the plasmid pSACII1 carrying the genes of restriction-modification system Eco29kI (isoschizomer of SacII) was determined. The cloning of the Eco29kI endonuclease and methylase genes into the plasmid vector pUC129 produced recombinant strain Escherichia coli K802[pECO29A15] with Eco29kI synthesis level about 100 times higher than in the parent strain. The restriction endonuclease was purified from Escherichia coli K802 [pECO29A15] cells to near homogeneity using column chromatography sequentially on phosphocellulose, hydroxyapatite, and heparin-Sepharose and rechromatography on phosphocellulose. Biochemical characterization of the homogeneous R Eco29kI is given. The enzyme has molecular mass 24.5 kD and is present in the solution as a monomer.
Pertzev
A V
AV
Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Moscow Region, Russia.
Kravetz
A N
AN
Mayorov
S G
SG
Zakharova
M V
MV
Solonin
A S
AS
D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D015183:Restriction Mapping
C035337:CCGCGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific