Lysonase Bioprocessing Reagent is an optimized, ready-to-use blend of rLysozyme™ Solution and Benzonase® Nuclease. rLysozyme Solution (see Technical Bulletin 334) is a highly purified and stabilized recombinant lysozyme with a specific activity 250 times greater than that of chicken egg white lysozyme (1). Benzonase Nuclease (see Technical Bulletin 261) is a genetically engineered non-specific endonuclease from Serratia marcescens (2, 3) that degrades all forms of DNA and RNA (single stranded, double stranded, circular, linear), reducing protein extract viscosity, and increasing protein yield (4). The combined activities of rLysozyme and Benzonase Nuclease significantly increase protein extraction efficiency and overall protein yield. Lysonase facilitates downstream processing of protein extracts in procedures such as chromatography,ultrafiltration, robotic liquid handling, and electrophoresis. This protocol describesE. coli lysis with Lysonase Bioprocessing Reagent treatment combined with a freeze/thaw of the cell pellet. Protocols for E. coli lysis using Lysonase with mechanical lysis, BugBuster®, and PopCulture™ Protein Extraction Reagents are described in Techncial Bulletins 054 (His•Bind® Kits), 245 (BugBuster Protein Extraction Reagent) and 323 (PopCulture Reagent). PopCulture extraction using Lysonase and purification of His•Tag® of GST•Tag™ fusion proteins in a 96-well format can be achieved with RoboPop™ Purification Kits (see Technical Bulletins 327 and 346). PopCulture extraction using Lysonase is also utilized in the RoboPop Solubility Screening Kit for high throughput (HT) screening of solubility optimization experiments (see Techncial Bulletin 362).