[NEB] NEBExpress® T4 Lysozyme

NEBExpress^® T4 Lysozyme은 박테리아 세포벽을 분해하는 재조합 펩티도글리칸 (murein) 가수분해 효소로 proteins, membrane proteins, DNA, RNA, or metabolites 추출 과정에 사용할 수 있습니다.

– 원핵세포의 peptidoglycan에서 N-acetylmuramic acid와 N-acetylglucosamine 사이의 β-1,4 결합을 가수분해

– Source: An E. coli strain that carries the bacteriophage T4 lysozyme gene

– NEBExpress T4 Lysozyme은 chicken egg white lysozyme 보다 활성이 200배 높음

Lysis reactions are scalable and compatible with high throughput workflows.
Fast and non-mechanical bacterial lysis; the lysate is ready to use and compatible with affinity resins.
Small (200 µg) and large (5 x 200 µg) formats are suitable for lysing 7 or 35 grams of cells, respectively.
Recombinant and animal free.

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.

Q&A

How are NEBExpress® T4 Lysozyme and chicken egg white lysozyme different?

NEBExpress^® T4 Lysozyme is a recombinant protein ~200 fold more active than chicken egg white lysozyme when used under the same conditions. Consequently, much less T4 Lysozyme (~ 1 ug of enzyme per 1 mL of cells) is needed to lyse E. coli in Tris buffer, versus 0.1 to 0.5 mg per 1 mL of chicken egg white lysozyme.

Can I use NEBExpress® T4 Lysozyme for DNA or RNA extraction?

Yes. NEBExpress^® T4 Lysozyme has no detectable endonuclease, exonuclease, RNase, or DNase activity and is therefore recommended for DNA and RNA extraction.

What buffers are compatible with NEBExpress® T4 Lysozyme?

NEBExpress® T4 Lysozyme is compatible with Tris-HCl, HEPES, and sodium phosphate-based buffers. It has an optimal pH range of 7.5-8.5 and can be supplemented with <25 mM NaCl, <2 mM EDTA, <1 mM MgCl₂, and Tween 20 or Triton X-100 <2%. The enzyme is also compatible with reducing agents such as DTT, β-mercaptoethanol, TCEP, and THP.

For optimal T4 Lysozyme activity, use either:

1 µL NEBExpress T4 Lysozyme (1 µg of enzyme) per 1 mL of resuspended cells in 50 mM Tris-HCl pH 7.5, 0.5 mM EDTA.
0.1 µL NEBExpress T4 Lysozyme per 1 mL of resuspended cells in NEBExpress® E. coli lysis reagent (NEB #P8116S).

Note that ≥ 2mM EDTA, > 25 mM NaCl, > 1 mM MgCl2 will start inhibiting enzyme activity, with a 50% loss of activity at 5 mM EDTA, 80% loss at 50 mM NaCl and 80% loss at 5 mM MgCl₂.

Tools & Resources

Protein Expression and Purification Selection Chart

Application	Kit	Advantages
High-Yield Expression	NEBExpress MBP Fusion and Purification System	Substantial yields (up to 100 mg/L) in more than 75% cases tested; uses the strong Ptac promoter.
	K. lactis Protein Expression Kit	Uses the strong LAC4 promoter; multiple integrations of plasmid results in higher yield.
	IMPACT™ Kit	Uses the T7 promoter for high level regulated expression.
	NEBExpress Cell-free E. coli Protein Synthesis System	Uses a highly active cell extract and T7 RNA Polymerase promoter to routinely achieve yields of 0.5 mg/ml.
Cell-Free Expression	NEBExpress Cell-free E. coli Protein Synthesis System	Quickly generates analytical amounts of protein from DNA plasmids or linear templates; amenable to scale-up
Cell-Free Expression	PURExpress® In Vitro Protein Synthesis Kit	Quickly generates analytical amounts of protein from DNA plasmids or linear templates
Co-Expression of Multiple Proteins	K. lactis Protein Expression Kit	Easily co-express 2–4 proteins.
Co-Expression of Multiple Proteins	PURExpress® In Vitro Protein Synthesis Kit	Bicistronic constructs or multiple plasmids with appropriate transcription and translation regulatory elements can be used.
Enhanced Solubility	NEBExpress MBP Fusion and Purification System	Fusion to MBP enhances solubility of proteins in E. coli.*
Enhanced Solubility	K. lactis Protein Expression Kit	Utilizes K. lactis eukaryotic folding pathway.
Affinity Tag Chromatography	IMPACT™ Kit	Utilizes an intein-chitin binding domain (CBD) tag on either the N- or C- terminus, offering single-step purification.
	NEBExpress MBP Fusion and Purification System	Fusion to MBP allows for purification on amylose resin.
	K. lactis Protein Expression Kit	Vectors are sold separately that generate fusions to MBP allowing for purification on amylose resin.
	NEBExpress Cell-free E. coli Protein Synthesis System	Compatible with all types of affinity purification; target protein can be synthesized from any T7 promoter vector
Post-translational Modification	K. lactis Protein Expression Kit	Secretion of both N- and O- glycosylated proteins.
Secreted Expression	K. lactis Protein Expression Kit	Eliminates cell lysis step, simplifying purification.
Toxic Proteins	K. lactis Protein Expression Kit	Secretion of protein from the cell.
Toxic Proteins	IMPACT™ Kit	Can express the toxic gene in two pieces and ligate proteins together using intein mediated protein ligation (IPL).