Crude protein extraction protocol for phage N15 protelomerase in vitro enzymatic assays

The phage N15 protelomerase enzyme (TelN) is essential for the replication of its genome by resolution of its telRL domain, located within a telomerase occupancy site (tos), into hairpin telomeres. Isolation of TeIN for in vitro processing of tos, however, is a highly complex process, requiring multiple purification steps. In this study a simplified protocol for crude total protein extraction is described that retains the tos-cleaving activity of TeIN for at least 4 weeks, greatly simplifying in vitro testing of its activity. This protocol may be extended for functional analysis of other phage and bacterial proteins, particularly DNA-processing enzymes. (C) 2011 Elsevier Inc. All rights reserved.