Novel modular type II restriction endonuclease, cspci, and the use of modular endonucleases for generating endonucleases with new specificities
AssigneeNew England Biolabs, Inc.
InventorsRichard Morgan, Geoffrey Wilson, Keith Lunnen, Daniel Heiter, Jack Benner, Celine Nkenfou, Stephen Picone
Life of this patent
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Abstract
A novel restriction endonuclease and methods of making the same are obtainable from either Citrobacter species 2144 (NEB#1398) or the recombinant stain Escherichia coli (NEB#1554) which cleaves at nt sequence 5′-CAANNNNNGTGG-3′ (SEQ ID NO:14) in double-stranded DNA molecules. The novel restriction endonuclease is a modular protein in which the specificity moiety is an independent module from the restriction-modification module.
Claims15
1-14. (canceled)
A method of making a Type II restriction endonuclease having an altered specificity;
- comprising:
- (a) selecting a restriction endonuclease characterized by a modular structure having a specificity subunit and a catalytic subunit, the specificity subunit further comprising an N-terminal domain for binding one half site of a bipartite recognition sequence and a C-terminal domain for binding a second half site of the bipartite recognition sequence;
- (b) modifying the specificity subunit;
- and
- (c) obtaining the Type II restriction endonuclease with altered specificity.
A substantially pure Type IIG restriction endonuclease obtainable from Citrobacter species 2144 (NEB#1398) (ATCC Patent Accession No. PTA-5846) or from Escherichia coli NEB#1554 (ATCC Patent Accession No. PTA-5887) capable of recognizing at least one sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, and cleaving the DNA on both sides of the recognition sequence.
An isolated DNA encoding CstMI restriction endonuclease obtainable from Escherichia coli NEB#1554 (ATCC Patent Accession No. PTA-5887) or from Citrobacter species 2144 (NEB#1398) (ATCC Patent Accession No. PTA-5846).
Isolated DNA encoding the restriction endonuclease of claim 1, wherein the DNA comprises a first DNA segment encoding an endonuclease and methyl transferase catalytic function and a second DNA segment encoding a sequence specificity function of the restriction endonuclease wherein the first and second DNA segments comprise one or more DNA molecules.
A recombinant DNA vector, comprising:
- at least one of a first DNA segment coding for the restriction and modification domains of CspCI restriction endonuclease and a second segment coding for the specificity domain of the restriction endonuclease.
A host cell transformed with a first DNA segment coding for the restriction and modification domains of CspCI restriction endonuclease and a second segment coding for the specificity domain of the restriction endonuclease wherein the first DNA segment and the second DNA segment are contained within one or more DNA vectors.
Dependencies are inferred from the claim text.