GONUTS has been updated to MW1.31 Most things seem to be working but be sure to report problems.
PMID:2269300
| Citation |
Kosak, HG and Kemper, BW (1990) Large-scale preparation of T4 endonuclease VII from over-expressing bacteria. Eur. J. Biochem. 194:779-84 |
|---|---|
| Abstract |
Endonuclease VII is the product of gene 49 of phage T4 and was the first enzyme shown to resolve Holliday structures in vitro [Mizuuchi, K. et al. (1982) Cell 29, 357-365]. Low amounts of the enzyme were originally purified from phage-infected cells [Kemper, B. & Garabett, M. (1981) Eur. J. Biochem. 115, 123-131]. We now report a purification procedure for milligram amounts of cloned endonuclease VII expressed in Escherichia coli with gene 49 under the control of a temperature-inducible promoter on a plasmid system [Tomaschewski, J. (1988) PhD Thesis, University of Bochum, FRG]. The protein was purified 500-fold from crude extracts in five steps with a recovery of 15%. The steps include (a) poly(ethyleneglycol)/dextran two-phase separation; (b) DEAE-cellulose; (c) single-stranded DNA-agarose; (d) Mono-Q and (e) Mono-S chromatography. The final protein was more than 98% pure as estimated from SDS/PAGE analysis. The protein has an apparent molecular mass of 17.8 kDa on SDS-containing polyacrylamide gels and 36 kDa when determined by gel filtration or sedimentation through sucrose gradients in the presence of high salt (600 mM NaCl). In the absence of additional salt, the enzyme has a tendency to aggregate and products of molecular masses differing in steps of about 18 kDa appear on SDS-containing polyacrylamide gels. |
| Links | |
| Keywords |
Blotting, Western; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Endodeoxyribonucleases/isolation & purification; Endodeoxyribonucleases/metabolism; Escherichia coli/genetics; Escherichia coli/growth & development; Escherichia coli/metabolism; Gene Expression; Genes, Viral; Molecular Weight; Plasmids; Substrate Specificity; T-Phages/enzymology; T-Phages/genetics; Transformation, Genetic |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0004519: endonuclease activity |
ECO:0000314: |
F |
The enzyme endonuclease VII cleaves the DNA into smaller pieces. This article presents a figure (3a and 3b) that shows the enzyme activity of endonuclease VIIcl (cl = clone, Y-axis) versus the sedimentation centrifugation fraction (including inset with fraction for molecular masses of each marker protein used). The activity of the endnuclease VIIcl enzyme is shown to be in between the appropriate masses of each protein (monomer and aggregated forms) used in the sedimentation experiment. |
complete | ||||
Notes
See also
References
See Help:References for how to manage references in GONUTS.
