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US 2016/0102306 A1Published application
High Fidelity Restriction Endonucleases
AssigneeNew England Biolabs, Inc.
InventorsZhenyu Zhu, Aine Quimby, Shuang-Yong Xu, Shengxi Guan, Hua Wei, Penghua Zhang, Dapeng Sun, Siu-hong Chan
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Abstract
Compositions and methods are provided for enzymes with altered properties that involve a systematic approach to mutagenesis and a screening assay that permits selection of the desired proteins. Embodiments of the method are particularly suited for modifying specific properties of restriction endonucleases such as star activity. The compositions includes restriction endonucleases with reduced star activity as defined by an overall fidelity index improvement factor.
Claims5
-18. (canceled)
A screening method comprising:
- i. identifying which amino acid residues in an amino acid sequence of a restriction endonuclease having star activity are charged amino acids;
- ii. mutating one or more codons encoding one or more of the charged residues in a gene sequence encoding the restriction endonuclease;
- iii. generating a library of gene sequences having one or more different codon mutations in different charged residues;
- iv. obtaining a set of proteins expressed by the mutated gene sequences;
- and
- v. determining a cleavage activity for each protein, and determining a fidelity index for each protein in a predetermined buffer, wherein determining a fidelity index comprises determining the ratio of the highest concentration of protein that does not show star activity to the lowest concentration of protein that completes digestion of the substrate.
3 more elements
Dependencies are inferred from the claim text.