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Mempro™ Cell-Free Protein Production in E. Coli

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Creative Biolabs can provide stable and high-quality Mempro™ cell-free protein production based on E. coli  lysates. We can accomplish your project perfectly according to your specific demands.

Conventional in vivo membrane protein prodction systems are often limited by host cell toxicity, low yield and in vitro purification using detergents. To overcome these limitations, we present an improved cell-free protein synthesis system based on E.coli lysates, which produces active and high-yield membrane proteins without the need of detergents or refolding stpes (Figure 1). This cell-free prodcution system, needed for amino acid incorporation in the presence of soluble tRNA and ribosome, activates an E. coli-derived cell extract for transcription and translation. We have direct and efficient control of the reaction environment due to the in vitro membrane protein synthesis is not enclosed behind a cell wall.

Mempro™ Cell-Free Protein Production in <em>E. Coli</em> Figure 1. Schematic of phosphoprotein synthesis from E. coli lysate. Sep-OTS: genetically encoded phosphoserine; CFPS: cell-free protein synthesis; NTPs: nucleoside triphosphates; lysis: breaking down cell membrane (Nature Communications, 2015)

Creative Biolabs selects the common S30 extract from an E. coli B strain (SL119), which is deficient in OmpT endoproteinase, ion protease and exonuclease V (recBCD). The S30 extract contains most enzymes necessary for protein synthesis, and from which endogenous mRNA as well as all low-molecular-weight compounds are removed to suppress background expression. Reactions with E. coli S30 extract are routinely set up as coupled transcription/translation systems. Not only plasmid DNA but also linear DNA from PCR reactions can be used as the templates. We also provide preparation of E. coli inner membrane vesicles, which can supply membrane-bound components and the lipid environment required for insertion and folding of synthetic membrane protein.

Mempro™ membrane protein production in E. coli has a large of advantages, including:

E. coli extract-based coupled transcription-translation cell-free system has been developed for large-scale production of protein samples for both X-ray crystallography (selenomethionine substitution) and NMR (stable-isotope labeling). For both cases, higher labeling/substitution efficiency can be achieved compared with the production using cell-based expression system. In addition, as the system is easily adapted to automated and/or high-throughput procedures, it is an especially suitable protein expression method for structural genomics and proteomics project.

MemproTM cell-free protein production in E. coli cannot produce specific eukaryotic folding proteins, therefore, Creative Biolabs provides Mempro™ cell-free protein production in wheat germ to address the potential problem. We can also provide other various Mempro™ membrane protein production services. Please feel free to contact us for a detailed quote.

References:
D. Schwarz, et al. (2008). Production of membrane proteins using cell-free expression systems. Proteomics, 8(19):3933-3946.
J. J. Wuu, et al. (2008). High yield cell-free production of integral membrane proteins without refolding or detergents. Biochim. Biophys. Acta., 1778(5): 1237-1250.
P. Javin, et al. (2015). Robust production of recombinant phosphoproteins using cell-free protein synthesis. Nature Communications, 6: 8168.




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