EXtracellular Plasmid RESource (EXPRESs) Consortium
EXPRESs is a collection of mammalian expression plasmids that encode the extracellular regions of cell surface receptor and secreted proteins as soluble recombinant molecules. The proteins are typically used in systematic protein interaction screens or cellular assays to identify novel interactions or pathways that are important in cellular recognition processes.
Laboratories interested in these approaches have created this resource to facilitate open and free exchange of these expression plasmids. It is hoped that this will encourage other groups to join our community and contribute their own plasmids in the future.
AVEXIS:
One popular method to detect interactions is AVEXIS (for Avidity-based extracellular interaction screen) in which the extracellular regions of receptor proteins are expressed as both monomeric baits and multimerised enzyme-tagged preys.
Ectodomain inserts:
The inserts that encode the whole ectodomains of cell surface receptors are flanked by two 8 bp cutting enzymes: NotI at the 5' end and AscI at the 3' end. These inserts can then be sub-cloned into a series of expression vectors which have different C-terminal tags. Most vectors are based on the pTT3 plasmid backbone which is described by Durocher et al. Nucleic Acids Research 2002 Jan 15;30(2):E9
A list of commonly-used bait and prey plasmids can be found here:
| Plasmid Type | Plasmid Name |
|---|---|
| Bait | Cd4d3+4-bio |
| Prey | Cd4d3+4-COMP-b-lac |
| His-tag | Cd4d3+4-6his |
| Bio-linker-his | Cd4d3-bio-linker-6His* |
| FLAG prey | CD200-Cd4d3+4-COMP-blac-3xFLAG-6his |
| Cd4d3+4-pentamer | BasiginCd4d3+4-COMP-his-stop |
| Type II expression | CD69-His-bio |
| Multi TM expression | CD36-bio-His |
Plasmid collections exist for:
- Basic AVEXIS: A list of all you need to get started with AVEXIS, including positive and negative controls
- Zebrafish receptors
- Zebrafish antibody plasmids
- P. falciparum merozoite surface protein plasmids
- P. vivax merozoite protein plasmids
- Human platelet receptors
- Human erythrocyte
- Babesia microti blood stage proteins
- P. falciparum sporozoite cell surface and secreted proteins
- Mouse sperm
- Mouse egg
Laboratories using AVEXIS:
- Christian Sollner, Max Planck Institute
- Mitch Levesque University of Zürich
- Gavin Wright, Wellcome Sanger Institute
Protocols:
Video showing how AVEXIS works with a detailed protocol description:
- Avidity based Extracellular Interaction Screening (AVEXIS) for the scalable detection of low affinity extracellular receptor-ligand interactions.Kerr SJ and Wright GJ. J. Vis. Exp. (61), e3881, DOI: 10.3791/3881 (2012).
Papers showing how AVEXIS can be used:
- Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC and Wright GJ Nature 2011 Nov 9; 480(7378):534-7.
- Construction of a large extracellular protein interaction network and its resolution by spatiotemporal expression profiling.Martin S, Söllner C, Charoensawan V, Adryan B, Thisse B, Thisse C, Teichmann S and Wright GJ Molecular & cellular proteomics 2010 Dec; 9(12):2654-65.
- A cell surface interaction network of neural leucine-rich repeat receptors.Söllner C and Wright GJ. Genome biology 2009; 10(9):R99.
- Large-scale screening for novel low-affinity extracellular protein interactions.Bushell KM, Söllner C, Schuster-Boeckler B, Bateman A and Wright GJ. Genome research 2008 Apr; 18(4):622-30.
- A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions.Sun Y, Gallagher-Jones M, Barker C, Wright GJ. Analytical Biochem. 2012 May 1;424(1):45-53.