Mempro™ Cell-Free C1 Domain-Containing Protein Production

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Creative Biostructure can provide unrivalled costom Mempro™ protein production services for C1 domain-containing proteins based on cell-free expression system. We can guarantee preserving the native conformation of C1 domain-containing proteins.

Mempro™ cell-free protein production system is the innovative system to produce integral membrane proteins, which bypasses the limitations that encountered in cell-based protein production system, such as host cell toxicity, low yield and solubilization and purification using detergents. Cysteine-rich C1 domains (also termed as phorbol esters/diacylglycerol binding domains) are involved in recruiting of proteins to the membrane. C1 domains bind an important sencondary messenger diacylglycerol (DAG) and the analogous phorbol esters, which can directly stimulate protein kinase C (PKC). A large group of signaling families, such as PKDs, chimaerins, RasGRPs, and diacylglycerol kinases (DGKs), possess the C1 domains.

Figure 1. Schematic of biological function of RasGRP1. (Elife, 2013)

Creative Biostructure has extensive experience in high-quality C1 domain-containing protein production using cell-free protein expression system, we can provide various strategies for Mempro™ cell-free protein production, including:

Mempro™ Cell-Free Protein Production in E. coli;
Mempro™ Cell-Free Protein Production in Wheat Germ;
Mempro™ Cell-Free Protein Production in Rabbit Reticulocyte;
Mempro™ Cell-Free Protein Production in Baculovirus.

Among Mempro™ cell-free membrane protein production systems, E. coli S30 extract is widely used for C1 domain-containing protein production. Wheat germ extract can provide eukaryotic translation system for C1 domain-containing protein expression. Baculovirus expression system from Autographa californica nucleopolyhedrovirus (AcNPV) is also a major system for C1 domain-containing protein expression. Most notably, rabbit reticulocyte lysate is another powerful tool for eukaryotic cell-free protein expression. To choose an appropriate source of the extract, many factors, such as the availability of materials, protein origin and complexity, downstream processing should be considered.

With the Mempro™ cell-free protein production platform, Creative Biostructure are capable of expressing, purifying and crystallizing C1 domain-containing proteins to perform further structural and functional analyses. Mempro™ cell-free membrane protein production has a wide range of advantages, including：

Very high yield;
Low cost in time and money;
Lack of toxicity;
Incorporation of non-natural amino acids into protein structure;
Relatively tolerant of additives.

Additionally, one challenge associated with cell-free protein production is that degradation of the DNA by endogenous nucleases. Creative Biostructure can optimize Mempro™ cell-free protein production for C1 domain-containing protein using plasmids due to they are circular and have no end for the exonucleases to attach.

We provide other various Mempro™ membrane protein production services. Please feel free to contact us for a detailed quote.

References:
F. Bernhard, et al. (2013). Cell-free expression - making a mark. Cur. Opin. Struct. Biol., 23: 374-380.
F. Colón-González and M. G. Kazanietz (2006). C1 domains exposed: from diacylglycerol binding to protein-protein interactions. Biochim. Biophys. Acta., 1761(8): 827-837.
J. Das and G. M. Rahman (2014). C1 domains: structure and ligand-binding properties. Chem. Rev., 114 (24): 12108–12131.
J. S. Iwig, et al. (2013). Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1. Elife, 2: e00813.