Creative Biostructure provides custom Mempro™ protein production services for P-loop containing nucleoside triphosphate (NTP) hydrolases using cell-free expression system. Our cell-free expression systems can best preserve the native conformation of P-loop containing nucleoside triphosphate hydrolases.
Mempro™ cell-free protein production system is the innovative platform to produce integral membrane proteins, which can bypass the limitations that encountered in cell-based protein production system, such as host cell toxicity, low yield and solubilization and purification using detergents. P-loop containing NTP hydrolase superfamily, which contains two distinct motifs, the Walker A motif (the P-loop proper) and Walker B motif that binds, respectively, the beta and gamma phosphate moieties of the bound NTP, and a Mg2+ cation. Proteins from this superfamily share a P-loop NTP hydrolase fold, which is the most common domain of the several distinct nucleotide-binding protein folds. The biochemical reaction catalysed the P-loop NTP hydrolase fold is the hydrolysis of the beta-gamma phosphate bond of a bound NTP.
Figure 1. The structural model of EH domain-containing protein 2, a member of P-loop containing NTP hydrolase superfamily. (OPM database)
Creative Biostructure has entensive experience in high-yield P-loop containing NTP hydrolase production using cell-free protein expression systems, we can provide various strategies for Mempro™ cell-free protein production, including:
Among Mempro™ cell-free membrane protein production systems, E. coli S30 extract is commonly employed for P-loop containing NTP hydrolase production. Wheat germ extract can provide eukaryotic translation system for P-loop containing NTP hydrolase expression. Baculovirus expression system from Autographa californica nucleopolyhedrovirus (AcNPV) is also a major system for P-loop containing NTP hydrolase expression, which is developed in the early stage that transfected insect cells in combination with vectors derived from the baculovirus. Crude rabbit reticulocyte lysate is also the most widely used for eukaryotic cell-free membrane protein expression. To employ an appropriate source of the extract, many factors, such as the availability of materials, protein origin and complexity, downstream processing, etc., should be considered.
With the Mempro™ cell-free protein production platform, Creative Biostructure is capable of expressing, purifying and crystallizing P-loop containing NTP hydrolases to facilitate the study of their biological functions.
Most notably, One challenge associated with Mempro™ cell-free membrane protein production is that degradation of the DNA by endogenous nucleases. Creative Biostructure can optimize Mempro™ cell-free membrane protein production for P-loop containing NTP hydrolases using plasmids owing 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.
Cell-free protein synthesis. (https://en.wikipedia.org/wiki/Cell-free_protein_synthesis)
F. Bernhard, et al. (2013). Cell-free expression - making a mark. Cur. Opin. Struct. Biol., 23: 374-380.
Y. Kawamura, et al. (2003). Systematic analyses of P-loop containing nucleotide triphosphate hydrolase superfamily based on sequence, structure and function. Genome Informatics, 14: 581-582.