Purification and crystallization of the EcoRV restriction endonuclease.
نویسندگان
چکیده
منابع مشابه
Fluorescent substrates for the EcoRV restriction endonuclease.
In the presence of Mg2+, the EcoRV restriction endonuclease cleaves DNA specifically at the sequence GAT'ATC (where denotes the point of scission) [ I ] . Even the change of a single base pair within this sequence will lead to a million fold reduction of EcoRV activity [2]. Paradoxically, gel retardation experiments in the absence of Mg2+ show that, unlike EcoRl and many other type 11 restricti...
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Ooryrprvcterium equii produces a new restriction endonuclease, an iso-schizemer of EcoKV (1). The bacterial strain does not contain significant levels of other nucleases, so the restriction endonuclease activity can be detected in crude extracts. Ihe enzyme is purified by ammonium sulphate fractionation (50-70 % saturation) and DEAE cellulose chranatography (elutes with 200 mM KC1 in PC buffer)...
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The type II restriction endonuclease BamHI has been expressed in E. coli, producing 100-fold more enzyme than the wild type Bacillus amyloliquefaciens H strain. This high yield has facilitated purification to homogeneity of large amounts of the enzyme, along with its crystallization in a form which diffracts to at least 1.9 A in X-ray analysis.
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When a plasmid containing restriction-modification (R-M) genes enters a naïve host, unmodified host DNA can be destroyed by restriction endonuclease. Therefore, expression of R-M genes must be regulated to ensure that enough methyltransferase is produced and that host DNA is methylated before the endonuclease synthesis begins. In several R-M systems, specialized Control (C) proteins coordinate ...
متن کاملCrystallization and preliminary X-ray analysis of restriction endonuclease FokI bound to DNA.
FokI is a type IIs restriction endonuclease which recognizes an asymmetric DNA sequence and cleaves DNA a short distance away from the sequence. The enzyme is bipartite in nature with its DNA recognition and cleavage functions located on distinct domains. We report here cocrystals of the complete FokI enzyme (579 amino acids) bound to a 20-bp DNA fragment containing its recognition sequence. Th...
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ژورنال
عنوان ژورنال: Journal of Biological Chemistry
سال: 1985
ISSN: 0021-9258
DOI: 10.1016/s0021-9258(18)89499-6