Cloned NsiI restriction-modification system
AssigneeLife Technologies Corporation
InventorsMary C. Longo, Michael D. Smith
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Abstract
The present invention discloses the cloning and expression in Escherichia coli of the NsiI restriction-modification system from Neisseria sicca, utilizing a two step protocol. Initial protection of the E. coli host with methylase expressed on a plasmid was required to stabilize a compatible plasmid carrying the endonuclease gene on a single DNA fragment. A chromosomal map was generated localizing the genes for NsiI methylase and endonuclease. An E. coli strain was constructed which produced high levels of NsiI endonuclease.
Claims15
A method of producing NsiI restriction endonuclease comprising:
- (a) growing a recombinant host cell which is protected from NsiI cleavage and which comprises a structural gene coding for the NsiI restriction endonuclease under conditions suitable for the expression of said endonuclease;
- and
- (b) isolating said NsiI endonuclease.
A method of using a vector comprising a structural gene coding for the NsiI restriction endonuclease, to prepare an NsiI restriction endonuclease, said method comprising:
- (a) introducing said vector into a host cell to produce a recombinant host cell;
- (b) culturing said recombinant host cell;
- and
- (c) isolating said restriction endonuclease from said recombinant host cell .
Dependencies are inferred from the claim text.