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| Protein Extraction & Process Improvement | | |  | Lysonase™ Bioprocessing Reagent
Lysonase™ Bioprocessing Reagent is an optimized, ready-to-use blend of rLysozyme Solution and Benzonase Nuclease. rLysozyme Solution contains a highly purified and stabilized recombinant lysozyme with specific activity 250 times greater than that of chicken egg white lysozyme. Benzonase Nuclease is a genetically engineered non-specific endonuclease that degrades all forms of DNA and RNA (single stranded, double stranded, circular, linear), reducing protein extract viscosity, and increasing protein yield. The combined activities of rLysozyme and Benzonase Nuclease significantly increase protein extraction efficiency and facilitate downstream processing of protein extracts.
To improve parallel processing of multiple E. coli culture samples and increase protein yields, use Lysonase Bioprocessing Reagent with PopCulture® Reagent, a concentrated mixture of specialized detergents that enables extraction and purification of recombinant proteins from E. coli directly from culture media without cell harvest, mechanical disruption, or extract clarification. For efficient protein extraction with BugBuster® Protein Extraction Reagent, use 10 ml Lysonase per 1 g cell paste. For efficient protein extraction with PopCulture Reagent, add 2 ml Lysonase per 1 ml extract. Store at –20°C.
rLysozyme™ Solution
rLysozyme Solution contains a highly purified recombinant lysozyme that can be used for lysis of E. coli. The enzyme catalyzes the hydrolysis of N-acetylmuramide linkages in bacterial cell walls. A small amount (40 U/ml) of rLysozyme enhances the efficiency of protein extraction with BugBuster® and PopCulture® Reagents. The specific activity of rLysozyme ( 1,700 KU/mg) for E.coli lysis is 250 times greater than that of egg white lysozyme. rLysozyme is optimally active at physiological pH. The product is provided as a ready-to-use solution at a concentration of 30 KU/µl in 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1NaCl, 0.1 mM EDTa, 1 mM DTT, and 0.1% Triton X-100. rLysozyme Solution is stable at –20°C.
rLysozyme™ Solution, Veggie™ Grade
rLysozyme Solution, Veggie Grade is a stabilized preparation of highly purified recombinant enzyme which has the same high specific activity as the enzyme in Novagen’s rLysozyme Solution, but is prepared in the absence of animal-derived products. From the growth and induction of cells to protein purification and storage, all steps in the production of this enzyme use only certified animal-free reagents. Because of its high specific activity (1,700 KU/mg), only a small quantity of Veggie Grade rLysozyme is required to break the cell wall of E. coli. Use the enzyme at 3–5,000 units/g cells to enhance protein recovery from cells treated with BugBuster® Protein Extraction Reagent, BugBuster HT, or PopCulture® Reagents, or treat the cells with the enzyme alone at a ratio of 45–60 KU/g cells. The product is supplied as a ready-to-use solution at a concentration of 30 KU/microliter in 50% Glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1 NaCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% Triton X-100. Solution is stable at –20° C.
Benzonase® Nuclease
Benzonase® Nuclease is a genetically engineered endonuclease from Serratia marcescens. It degrades all forms of DNA and RNA (single stranded, double stranded, linear and circular) while having no proteolytic activity. It is effective over a wide range of conditions and possesses an exceptionally high specific activity. The enzyme completely digests nucleic acids to 5′-monophosphate terminated oligonucleotides 2 to 5 bases in length (below the hybridization limit), which is ideal for removal of nucleic acids from recombinant proteins, enabling compliance with FDA guidelines for nucleic acid contamination. The ability of Benzonase to rapidly hydrolyze nucleic acids makes the enzyme an excellent choice for viscosity reduction to reduce processing time and increase yields of protein. For example, the enzyme is compatible with BugBuster® and PopCulture® Protein Extraction Reagents and can therefore be added along with these reagents to eliminate viscosity and remove nucleic acids from E. coli extracts.
The enzyme consists of two subunits of 30 kDa each. It is functional between pH 6 and 10 and from 0°C to 42°C and requires 1–2 mM Mg2+ for activation. The enzyme is also active in the presence of ionic and non-ionic detergents, reducing agents, PMSF (1 mM), EDTA (1 mM) and urea (relative activity depends on specific conditions). Activity is inhibited by > 150 mM monovalent cations, > 100 mM phosphate, > 100 mM ammonium sulfate, or > 100 mM guanidine HCl. Benzonase Nuclease is available in ultrapure (> 99% by SDS-PAGE) and pure (> 90%) grades. Both preparations are free of detectable protease and have specific activity > 1 × 106 units/mg protein. The > 99% purity grade is tested for endotoxins and contains < 0.25 EU/1,000 units. Supplied as a 0.2 m filtered solution in 50% glycerol. Store at –20°C.
Unit definition: one unit is defined as the amount of enzyme that causes a DA260 of 1.0 in 30 minutes, which corresponds to complete digestion of 37 mg DNA. |
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