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 ca rrying 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 ch romatography and sedimentation equilibrium analyses were used to show that the SbcC and SbcD polypeptides dissociate at some stage after pur ification and that this dissociation is reversed by the addition of Mn 2+. We demonstrate that SbcD has the potential to form a secondary str uctural motif found in a number of protein phosphatases and suggest th at it is a metalloprotein that contains the catalytic center of the Sb cCD exonuclease.