New England Biolabs, P0774S, NEBExpress® GamS Nuclease Inhibitor

NEBExpress GamS Nuclease Inhibitor is a recombinant protein that inhibits Exonuclease V (RecBCD) activity and stabilizes linear DNA templates in Related Categories NEBExpress, ®, Cell-free, E. coli, Protein Synthesis System,, Cell-Free Protein Expression,, Protein Expression Applications NEBExpress® Cell-free E. coli Protein Synthesis System,, Cell-Free Protein Expression,, Protein Expression Specification Storage Buffer 50 mM Tris-acetate pH 8.2 FAQ Q: How does NEBExpress® GamS Nuclease Inhibitor enhance protein yield? A: The NEBExpress Cell-free E. coli Protein Synthesis System is engineered to be compatible with linear DNA. However, protein yield produced from a linear DNA template is typically lower than the protein yield produced from plasmid DNA. This is because of the presence of an E. coli exonuclease, RecBCD (Exo V), which is responsible for degrading the linear DNA template. GamS can be added to the synthesis reaction to effectively inhibit the activity of RecBCD, so that the linear DNA can be stabilized for sustainable transcription. Q: How much linear DNA template and NEBExpress® GamS Nuclease Inhibitor should I add to my reaction? A: As a starting point, we suggest adding 250 ng of linear DNA and 1.5 μg GamS (1μL) in a 50 μL reaction to enhance protein yield. The size of the gene and the DNA construct can contribute to the stability of the linear DNA and the rate of transcription. Therefore, different protein targets may require further optimization to determine the best ratio of template and GamS. During the experimental setup, it is recommended to add the linear DNA template in the last step to allow GamS to bind and inhibit RecBCD before RecBCD has a chance to act on the DNA. In addition, the protein synthesis reaction can be scaled to larger volumes, in which case the amount of linear DNA and GamS should also be scaled proportional to the reaction volume. Q: Is more NEBExpress® GamS Nuclease Inhibitor always better? A: No! There is an optimal range of GamS in a cell-free protein synthesis reaction. In the NEBExpress Cell-free E. coli Protein Synthesis System, it’s determined to be ~2.4 μM, or 1.5 μg per 50 μL reaction. Adding more GamS can cause the protein yield to decrease. This is most likely because GamS is an acidic protein and acts as a DNA mimic. At high concentrations, it may interact with other enzymes and interfere with the transcription and translation process. Q: Can I freeze-thaw NEBExpress®GamS Nuclease Inhibitor? A: NEBExpress GamS Nuclease Inhibitor is stored in 100% aqueous buffer for optimal compatibility with protein synthesis reactions. It should be stored at -20˚C and freeze-thaw cycles should be avoided. If multiple freeze-thaws are anticipated, we recommend aliquoting the protein during the first usage. Q: How should I prepare my linear DNA? A: PCR kits such as NEB Q5® High-Fidelity PCR Kit (NEB #E0555) can produce excellent yield of linear DNA. Linear DNA should be purified prior to being used in a cell-free protein synthesis reaction. We recommend purification using the Monarch® PCR & DNA Cleanup Kit (5 µg) (NEB #T1030).