Overexpression, purification, and characterization of the SbcCD protein from Escherichia coli.
The sbcC and sbcD genes mediate palindrome inviability in Escherichia coli. The sbcCD operon has been cloned into the plasmid pTrc99A under the control of the strong trc promoter and introduced into a strain carrying a chromosomal deletion of sbcCD. The SbcC and SbcD polypeptides were overexpressed to 6% of total cell protein, and both polypeptides copurified in a four-step purification procedure. Purified SbcCD is a processive double-strand exonuclease that has an absolute requirement for Mn2+ and uses ATP as a preferred energy source. Gel filtration chromatography and sedimentation equilibrium analyses were used to show that the SbcC and SbcD polypeptides dissociate at some stage after purification and that this dissociation is reversed by the addition of Mn2+. We demonstrate that SbcD has the potential to form a secondary structural motif found in a number of protein phosphatases and suggest that it is a metalloprotein that contains the catalytic center of the SbcCD exonuclease.
Connelly
J C
JC
Institute of Cell and Molecular Biology, University of Edinburgh, Kings Buildings, Edinburgh EH9 3JR, United Kingdom.
de Leau
E S
ES
Okely
E A
EA
Leach
D R
DR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D002413:Cations, Divalent / Q000494:pharmacology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003851:Deoxyribonucleases / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005092:Exonucleases / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D009876:Operon; D017433:Protein Structure, Secondary; D016335:Zinc Fingers
D001426:Bacterial Proteins; D002413:Cations, Divalent; D029968:Escherichia coli Proteins; C061563:SbcC protein, Bacteria; C489229:SbcC protein, E coli; D008345:Manganese; D000255:Adenosine Triphosphate; D003851:Deoxyribonucleases; D005092:Exonucleases; C081229:sbcD protein, E coli