ABSTRACT
The gene of the new site-specific methyltransferase M.SscL1I belonging to the same modification-restriction system as the previously described by us site-specific endonuclease SscL1I has been cloned from the natural strain Staphylococcus sp. L1. A plasmid to express the methylase gene under control of the T7 phage-specific promotor has been constructed. Conditions were found to express the recombinant methylase M.SscL1I and to purify it to near homogeneity. It is shown that the methylase modifies the adenine base in the recognition site 5;-GANTC-3;.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/genetics , Staphylococcus/enzymology , Chromatography, Gel , Cloning, Molecular , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Deoxyribonucleases, Type II Site-Specific/metabolism , Electrophoresis, Polyacrylamide Gel , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
A site-specific endonuclease SscL1 I preparation has been isolated and purified to near homogeneity from the strain Staphylococcus sp. L1 without admixtures of other nuclease activity. DNA cleavage proceeds according to the scheme: 5'-G down arrow ANTC-3' 3'-CTNA up arrow G-5', and thus the isolated enzyme is an isoschizomer of restriction endonuclease HinfI and belongs to the second class of restriction endonucleases. SscL1 I works over a broad range of temperature and pH. The enzyme is characterized by high stability during storage.