myTXTL GamS Nuclease Inhibitor Protein is designed to enhance protein production in myTXTL Sigma 70 Master Mix when switching from circular to linear DNA templates in order to produce a recombinant protein.
By preventing DNA degradation resulting from exonuclease RecBCD activity, high-yield cell-free protein production with efficiencies comparable to yields from circular templates is readily achieved. This is especially relevant for protein and enzyme engineering projects with high sample throughput. In addition to myTXTL, GamS can also be advantageous for gene expression using other bacterial cell-free systems.
- Superior Performance – Reliably prevents degradation of linear DNA in the myTXTL system.
- Powerful – Instantly boosts protein yield from linear templates.
- Easy Handling – Just add an aliquot to the myTXTL Sigma 70 Master Mix.
- Maximum Flexibility – Works with linear and circular DNA templates.
- Versatile – Compatible with other bacterial cell-free expression systems.
Alternatively, the myTXTL Linear DNA Expression Kit and myTXTL T7 Expression Kit include the myTXTL Linear DNA Master Mix that has been further engineered to be amenable for linear DNA templates without additional stabilizers.
In applications with high sample throughput, such as protein and enzyme engineering, linear DNA input material for cell-free expression is desirable. Utilizing either PCR products or readily available gene fragments from a synthesis company, typically suitable for immediate use without any further purification, accelerates the validation process, thus the entire design-build-test-learn cycle.
The exonuclease complex RecBCD often present in E. coli-based, in vitro, cell-free expression systems is mainly responsible for template degradation of linear DNA, thereby lowering protein output. A popular strategy to enhance protein yield is based on inhibiting RecBCD’s enzymatic activity by a protein called GamS. Recombinantly expressed and purified in E. coli (>95 % confirmed by SDS-PAGE analysis), this short form of the natural protein Gam found in bacteriophage lambda (UniProt entry: P03702) enables protein production efficiencies at the level of reactions set up with plasmid DNA.
12 µL myTXTL reactions set up with 20 nM P70a-deGFP linear control fragment and myTXTL Sigma 70 Master Mix supplemented with 10 µM myTXTL GamS Nuclease Inhibitor Protein (expressed and purified from E. coli) were incubated at 29°C for 16 hours. Expressed enhanced green fluorescent protein was quantified according to the fluorescence signal from an eGFP standard curve (ex. 485 nm, em. 535 nm).
High yield from linear DNA templates
12 µL myTXTL reactions were set up with 20 nM P70a-deGFP linear control fragment and myTXTL Sigma 70 Master Mix supplemented with 10 µM myTXTL GamS nuclease inhibitor protein. In a black 384-well plate, enhanced green fluorescent protein-related fluorescence (ex. 485 nm, em. 535 nm) was recorded every 3 minutes at 29°C. Onset of fluorescence signal increase was detected after 12 min of incubation corresponding to expression of functional deGFP.
If you have started your project with plasmid templates for which you purchased the myTXTL Sigma 70 Master Mix, but now you want to try out linear DNA templates with the myTXTL Sigma 70 Master Mix you still have available in your lab. A simple addition of myTXTL GamS Protein to the myTXTL Sigma 70 Master Mix significantly boosts protein output from linear DNA templates in this master mix. For future work with linear DNA templates we would recommend the myTXTL Linear DNA Expression kit or myTXTL T7 Expression kit as both use the myTXTL Linear DNA Master Mix.
We always recommend keeping the thawing-and-freezing cycles as low as possible which might require aliquoting the original sample. However, myTXTL GamS Protein can be subjected to at least five thawing-and-freezing cycles.
For fastest sample throughput, PCR products can be directly used as template material for cell-free expression so long as the final glycerol concentration in the reaction is 0.1% or lower and the PCR reactions on average are making enough linear DNA to facilitate the protein yield needed. If a known fixed template concentration is preferred, PCR products should be subjected to a standard PCR clean up procedure with a final elution in molecular biology grade, nuclease-free water. This might also slightly boost the protein yield per reaction. Linear DNA can also be ordered and used directly in myTXTL from several commercial DNA suppliers. Please refer to our myTXTL Handbook and IDT App note for information about linear DNA template design requirements.
Yes, it is expected that protein yield resulting from linear templates is diminished compared to its circular plasmid version. A decrease of 10-30% is considered to be within the normal range.
10 mM Tris/HCl pH 7.5.
Utilizing linear DNA templates greatly increases the speed of the design-build-test-learn cycle as laborious steps like cloning, transformation and purification are no longer necessary. This is particularly useful when working with a high number of variants of a single protein that need to be studied and validated. The reduced costs of linear DNA can also expand the sampled sequence space for protein designs compared to the plasmid format.