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Crystallization and preliminary X‐ray characterization of two thermostable DNA nucleases

Crystallization and preliminary X‐ray characterization of two thermostable DNA nucleases Temperature‐tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA‐cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4‐methyl pentandiol, dioxane, ethanol or 2‐propanol as precipitants. The addition of a 10‐mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Crystallographica Section F Wiley

Crystallization and preliminary X‐ray characterization of two thermostable DNA nucleases

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References (15)

Publisher
Wiley
Copyright
Copyright © 2006 Wiley Subscription Services, Inc., A Wiley Company
ISSN
1744-3091
eISSN
1744-3091
DOI
10.1107/S1744309106050548
pmid
17142920
Publisher site
See Article on Publisher Site

Abstract

Temperature‐tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA‐cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4‐methyl pentandiol, dioxane, ethanol or 2‐propanol as precipitants. The addition of a 10‐mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes.

Journal

Acta Crystallographica Section FWiley

Published: Dec 1, 2006

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