The NEBExpress® Cell-free E. coli Protein Synthesis System is a 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.
The NEBExpress® Cell-free E. coli Protein Synthesis System contains all the components required for protein synthesis, except for the target template DNA. It is a combination of a highly active cell extract from a genetically engineered strain, a reaction buffer, and an optimized T7 RNA Polymerase, which together yield robust expression of a wide variety of protein targets ranging from 17 to 230 KDa.
Protein synthesis is achieved with a short incubation and 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, synthesized protein can be isolated from the reaction mixture using affinity purification techniques, such as immobilized metal affinity chromatography (IMAC), for further structural and/or functional characterization.
Figure 1: Protein synthesis 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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