Mempro™ Detergent-Free PH Domain-Like Protein Production

Creative Biostructure can provide high-quality custom Mempro™ pleckstrin homology (PH) domain-like protein production services using detergent-free expression system. Our skillful scientists can perform the production, purification and characterization of PH domain-like superfamily proteins.

PH domain-like proteins, a group of signaling proteins, which are recruited to target to the plasma membrane. Over 560 human membrane proteins contain PH domains, the significant feature of these PH domain is the binding of phosphatidylinositol phosphate (PIP) lipids with high affinity. PH domains share a common core structure, which is composed of two antiparallel β-sheets forming a β-sandwich (Figure 1). Owing to the structure of PH domain, it is difficult to obtain a purified PH domain-like proteins for structural and functioanl studies. Conventional approaches for purification of PH domain-like proteins need the form of detergent-solubilized samples. However, detergent can induce changes on membrane protein conformation. Stable solubilization and native conformation of PH domain-like proteins is therefore an important issue in biotechnology as well as in biochemistry and structural biology. Our alternative detergent-free expression systems can best preserve the native conformation of PH domain-like proteins for further structural studies.

Figure 1. Locations of relevant residues in the crystal structure of General Receptor for Phosphoinositides 1 (GRP1) PH domain. (Biochemistry, 2011)

Creative Biostructure has strong expertise in high-yield PH domain-like protein production using detergent-free membrane protein expression systmes, we can adopt various strategies for Mempro™ detergent-free PH domain-like protein production, including:

• Mempro™ PH domain-like protein production using nanodiscs

Nanodiscs are the self-assembly system to stabilize membrane proteins removed from the membrane by membrane scaffold proteins (MSPs) in no need of detergent. Creative Biostructure can reconstitute PH domain-like proteins into nanodiscs in detergent-free methodology: Expressing PH domain-like proteins in a cell-free system with the addition of pre-assembled nanodiscs to complete the solubilization step.

• Mempro™ PH domain-like protein production using amphipols

The amphipathic amphipols have the ability to “trap” around transmembrane regions of proteins, allowing them to stay folded. Creative Biostructure can apply A8-35 to solubilize PH domain-like proteins during purification step.

• Mempro™ PH domain-like protein production using poly (styrene-co-maleic acid) lipid particles (SMALPs)

The SMALPs are self-assembled by the simple addition of the SMA co-polymer. At neutral or alkaline pH, a disc-like structure assembles itself, encapsulating the PH domain-like proteins in a form amenable to be purified.

These novel detergent-free technologies for PH domain-like protein production can be obtained easily, and enabling more comprehensively structural and functional studies.

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

References:
C. Pilling, et al. (2011). GRP1 PH domain, like AKT1 PH domain, possesses a sentry glutamate residue essential for specific targeting to plasma membrane PI(3,4,5)P3. Biochemistry, 50(45): 9845-9856.