Creative Biostructure provides custom Mempro™ protein production services for pentameric ligand-gated ion channels (pLGICs) using cell-free expression system. Our patent cell-free expression systems can best preserve the native conformation of pLGICs.
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. The pLGICs belong to single neurotransmitter receptor superfamily, which can regulate fast synaptic communication by converting nerve signals into an electrical response. pLGICs are known as transmembrane ion channels that open to allow ions such as Na+, K+, Ca2+, or Cl− to pass through the membrane, which are composed of five homologous subunits. It is shown that theses subunits consist of at least two different domains, including a transmembrane domain which forms the channel gate and the ion selective filter, and an extracellular domain which includes the ligand binding location (an allosteric binding site). In addition, pLGICs are divided into three classes: Cys-loop receptors, Ionotropic glutamate receptors and ATP-gated channels.
Figure 1. A cartoon showing Neurotransmitter-gated ion-channel transmembrane region. (Wikipedia)
Creative Biostructure has entensive expertise in high-yield pLGICs 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 pLGICs production. Wheat germ extract can provide eukaryotic translation system for pLGICs expression. Baculovirus expression system from Autographa californica nucleopolyhedrovirus (AcNPV) is also a major system for pLGICs 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 utilized for eukaryotic cell-free transmembrane 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 pLGICs 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 pLGICs 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.J. A. Dent (2010). The evolution of pentameric ligand-gated ion channels. Adv. Exp. Med. Biol., 683: 11-23.L. Sauguet, et al. (2014). Crystal structures of a pentameric ligand-gated ion channel provide a mechanism for activation. Proc. Nat. Aca. Sci. U. S. A., 111(3): 966–971.