NEBExpress® T4 Lysozyme is a recombinant murein hydrolase that breaks down the bacterial cell wall by hydrolyzing the β-1,4 linkage between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan of prokaryotic cells (gram-negative and some gram-positive bacteria). It can be used to extract soluble proteins, membrane proteins, DNA, RNA, or metabolites.
Source: An E. coli strain that carries the bacteriophage T4 gene e.
Figure 1: NEBExpress T4 Lysozyme is 200-fold more active than chicken egg white lysozyme.
T7 Express E. coli expressing vGFP was lysed with NEBExpress T4 Lysozyme (T4L) or Chicken egg white lysozyme (ChL): 4 UOD600 of cell pellet were resuspended in 200 µL of 50 mM Tris-HCl pH 7.5 and lysed for 5 minutes at room temperature in the presence of NEBExpress T4 Lysozyme or Chicken egg white lysozyme at 0.1, 1, 10, 100 or 200 ug enzyme per 1 mL of cell suspension. The soluble proteins were harvested by centrifugation and analyzed on SDS-PAGE.
Figure 2: Optimal protein extraction with NEBExpress T4 Lysozyme in the presence of NEBExpress E. coli Lysis Reagent.
T7 Express E. coli expressing vGFP was lysed with NEBExpress T4 Lysozyme (T4L) in 50 mM Tris-HCl pH 7.5 or in NEBExpress E. coli Lysis Reagent (NEB #P8116S): 4 UOD600 of cell pellet were resuspended in 200 µL of Tris buffer or NEBExpress E. coli Lysis Reagent and lysed for 5 minutes at room temperature in the presence of NEBExpress T4 Lysozyme at 0, 0.1 or 1 ug per 1 mL of cell suspension. The soluble proteins were harvested by centrifugation and analyzed on SDS-PAGE.
This product is related to the following categories:
Tris, HEPES and sodium phosphate buffers ranging from pH 6.5 - 9.5; optimal pH is 7.5 - 8.5.
0.5 - 2 mM EDTA, which will improve lysis efficiency on gram negative bacteria. EDTA concentrations above 2mM start inhibiting enzyme activity, with a 50% loss at 5 mM EDTA.
≤ 25 mM NaCl; enzymatic activity is inhibited by 80% at 50 mM NaCl
≤ 1 mM MgCl2; enzymatic activity is inhibited by 80% at 5 mM MgCl2
≤ 2% Triton X-100 or Tween 20
Protease inhibitor cocktails (with ≤ 2 mM EDTA)
Reducing agent such as DTT, β-mercaptoethanol, TCEP and THP.
For optimal lysis performance, include NEBExpress E. coli Lysis Reagent (NEB #P8116S)
NEBExpress T4 Lysozyme can be added directly to resuspended cells or pre-mixed with the buffer prior to resuspension of the cell pellet.
Use 1 µL of NEBExpress T4 Lysozyme per 1 mL of cells resuspended at 10 UOD600. For example, for a 5 mL culture harvested at OD600 of 2, 10 UOD600 of cells are obtained (5 mL x 2 OD600 = 10 UOD600).
If weighing the cell pellet, use 30 µL of NEBExpress T4 Lysozyme per 1 gram of cell paste resuspended in 30 mL buffer.
The lysate may become viscous due to the release of DNA. For optimal lysis yield, eliminate viscosity with Micrococcal Nuclease (NEB #M0247). Add 1mM CaCl2 and 1 uL of Micrococcal Nuclease per 1 mL of lysate, mix and incubate for 5 minutes at room temperature.
Lysis efficiency can be strain dependent. For hard to lyse samples, use 5 to 15 µL of NEBExpress T4 Lysozyme per 1 mL of resuspended cells.
Cell pellets subjected to one freeze-thaw cycle prior to NEBExpress T4 Lysozyme treatment tend to lyse better.
References
Jensen, H.B., Kleppe K. (1972). Effect of Ionic Strength, pH, Amines and Divalent Cations on the Lytic Activity of T4 Lysozym. Eur. J. Biochem. 28, 116-122.
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Certificate of Analysis
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