Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space.

Nucleic Acids Research
Kyle JacobyAndrew M Scharenberg

Abstract

LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ≈ 20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering-many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our resul...Continue Reading

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Datasets Mentioned

BETA
BK008016

Methods Mentioned

BETA
targeted gene modification
immunoprecipitation
electrophoresis
flow cleavage assay
flow cytometry
affinity purification
affinity purification chromatography
size exclusion chromatography
protein folding
DNA cleavage assay

Software Mentioned

HKL2000
HjeMI
FloJo Star
REFMAC
MacVector
COOT ( Crystallography Object - Oriented Toolkit )
FloJo
Flowjo
/ CCP4i

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