NEBExpress® Cell-free E. coli Protein Synthesis System

Due to the tight coupling of transcription and translation in prokaryotic cells, bacterial lysate systems are most commonly used in cell-free protein synthesis (CFPS). Bacteria strains can be genetically engineered to yield optimal protein expression performance, and the lysate is often called “S30 extract” as early lysate preparation protocols used centrifugation at a specific sedimentation rate (Svedberg unit) to remove cellular debris. In cell extract-based systems, the necessary enzymes and factors for transcription and translation already exist in the lysate. Various energy sources, nucleotides and amino acids are added to the lysate or reaction buffer to enhance protein synthesis.

The NEBExpress® Cell-free E. coli Protein Synthesis System is a lysate-based coupled transcription/translation system designed to synthesize proteins encoded by a DNA template under the control of a T7 RNA Polymerase promoter. The system offers high expression levels, the ability to produce high molecular weight proteins, scalability, and is cost-effective for high throughput expression applications.  The speed and robustness of the system facilitates protein synthesis in applications such as protein engineering, mutagenesis studies and enzyme screening. In addition, it can be used to generate proteins for biophysical and structure-function analyses.

Protein synthesis is achieved using a short incubation and the synthesized protein is compatible with downstream purification or analysis by SDS-PAGE, Western Blot or direct functional assay. The novel formulation of this system allows samples to be loaded directly onto SDS-PAGE, without the need for acetone or TCA precipitation.  Additionally, the synthesized protein can be isolated from the reaction mixture by affinity purification techniques such as immobilized metal affinity chromatography (IMAC) for further structural and/or functional characterization. 
 

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Application Notes NEBExpress® Cell-free E. coli Protein Synthesis System
Protein Expression Using the NEBExpress Cell-free E. coli Protein Synthesis System
50 µL reactions containing 250 ng template DNA were incubated at 37°C for 3 hours. 2 µL of each reaction were analyzed by SDS-PAGE using a 10–20% Tris-glycine gel. The red dot indicates the protein of interest. M = Unstained Protein Standard, Broad range (NEB #P7717), “Neg” = negative control, no DNA. 
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