Large-scale preparation of T4 endonuclease VII from over-expressing bacteria.
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.
Kosak
H G
HG
Institut für Genetik der Universität zu Köln, Federal Republic of Germany.
Kemper
B W
BW
D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015870:Gene Expression; D005814:Genes, Viral; D008970:Molecular Weight; D010957:Plasmids; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic
D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII