Creative Biostructure has a long history of expressing recombinant proteins. Our scientists developed an advanced custom Mempro™ protein production services for phosphotyrosine protein phosphatases (PTPs) II using cell-free expression system. Our expression platform holds many advantages for membrane protein production, such as high yield, solubility, no host cell toxicity and so on.
Phosphotyrosine phosphatase plays a key role in many cellular processes, including cellular differentiation, cell growth, signal transduction, and so on. Coupled with tyrosine kinases, PTPs control the phosphorylation state of biological molecules. Based on the mechanism of catalysis, PTPs can be classified into four categories: the tyrosine-specific phosphatases, the VH1-like dual specificity phosphatases, the cdc25 phosphatases, and the low molecular weight phosphatases (LMW-PTPs). Class II only contains low-molecular-weight phosphotyrosine phosphatase (LMW-PTP). This class of enzyme acts on platelet-derived growth factor and insulin receptors for down regulation. The structure of LMW-PTP differentiates it from other PTPs while it has the same reaction mechanism and an active site sequence motif CXXXXXR. The crystal structure of a bovine liver PTP has been resolved. It consists of an α/β protein domain with a phosphate binding motif. Two α-helices located on both sides are connected by the four-stranded, open-twisted β-sheet. Cysteine 12 is a conserved residue for catalysis acting as a nucleophile. The sulphate group forms hydrogen bonds with the amide groups of the binding sites. In addition, the cysteine side chains interacts with the guanidinium group of arginine 18. Together with the hydrophobic residues on the edge of the phosphate binding sites, the cysteine residues define the substrate binding and the reaction mechanism of the LMW-PTP.
Figure 1. Structure of a low-molecular-weight phosphotyrosine protein phosphatase. (Wikipedia)
Creative Biostructure has a wealth of experience in high-yield PTP II production using cell-free protein expression systems, we can provide various strategies for Mempro™ cell-free protein production, including:
Among Mempro™ Cell-Free Protein Production systems, E. coli S30 extract system is the optimal system for LMW-PTP production. E. coli S30 extract system contains the translational machinery required for both prokaryotic and eukaryotic protein. It’s widely applied to a variety of membrane proteins for its fast and active production. Furthermore, using E. coli extract has less possibility of cross-reactivity or interference with the endogenous proteins in eukaryotic system.
With the Mempro™ cell-free protein production platform, Creative Biostructure provides phosphotyrosine protein phosphatases II in a cost-effective manner. We also provide protein crystallization and activity assay 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.Ago H, Kanaoka Y, Irikura D, et al. Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis[J]. Nature, 2007, 448(7153): 609-612.Hebert H, Jegerschöld C. The structure of membrane associated proteins in eicosanoid and glutathione metabolism as determined by electron crystallography[J]. Current opinion in structural biology, 2007, 17(4): 396-404.