myTXTL – Cell-Free Expression
myTXTL GamS Nuclease Inhibitor Protein
A powerful supplement to boost gene expression from linear DNA templates in myTXTL Cell-Free Expression system

myTXTL GamS Nuclease Inhibitor is a recombinant protein that stabilizes linear DNA templates used in combination with E. coli-based in vitro expression systems. 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 in particular relevant for protein and enzyme engineering projects with high sample throughput. 

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. It supports synthesis of the same great variety of proteins as the myTXTL Sigma 70 Master Mix alone. In addition, myTXTL GamS Protein can also be advantageous for gene expression using other bacterial cell-free systems. 

myTXTL GamS Nuclease Inhibitor Protein Delivers:

  • Superior Performance – Reliably prevents degradation of linear DNA in the myTXTL system
  • Powerful – Boots protein yield from linear templates instantly
  • 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 as well as the myTXTL T7 Expression kit include the myTXTL Linear DNA Master Mix that has been further engineered to be amenable for linear DNA templates without any additional stabilizers.

Ordering Information
Size
Cat#
Qty
Price
myTXTL GamS Protein
Single tube of purified GamS-His6 nuclease inhibitor protein at 150 µM, and volume sufficient for required reactions.
501024
$68
Available and in-stock for immediate shipment.
Additional Formats

Bulk offering available upon request.

Contact us for further information.



Overview

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.

GamS Protein Inhibition

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. 

 

Performance

Expression Kinetics

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.

FAQs
What is the recommended working concentration in a myTXTL reaction?

We recommend setting the myTXTL GamS Protein concentration in a myTXTL reaction at 10 uM (myTXTL GamS stock solution is provided at 150 uM).
You offer the myTXTL Linear DNA Master Mix optimized for gene expression from linear and circular templates. In which situation do I need myTXTL GamS Protein?

For example, 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 in those situations. Later on, you could potentially consider purchasing the myTXTL Linear DNA Expression kit or myTXTL T7 Expression kit as both list the myTXTL Linear DNA Master Mix.
What is in the myTXTL GamS Protein storage buffer?

10 mM Tris/HCl pH 7.5.
How often can I freeze and thaw GamS without losing activity?

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.
How should I prepare my linear template DNA and what are your design recommendations for linear DNA templates?

For fastest sample throughput, PCR products can be instantly utilized as template material for cell-free expression. 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. Please refer to our myTXTL Handbook for information about linear DNA template design requirements.
Is there a difference in protein yield when working with linear DNA templates?

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.
What is the advantage of working with linear DNA templates over circular DNA templates?

Utilizing linear DNA templates extremely increases the speed of the design-build-test-learn cycle as laborious steps like cloning, transformation as well plasmid preparation 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.

Please also refer to the FAQ section of the myTXTL Protein Expression Kits.

 

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