ESPRIT technology beta

lab equipment
Screening tens of thousands of expression constructs of a target gene. Constructs are made as a random library and printed on membranes for soluble expression analysis by hybridisation of fluorescent antibodies.

Procedure

To screen a target through ESPRIT involves active participation by a scientist working closely with the Hart laboratory. The scientist should be experienced in molecular biology techniques including DNA manipulation and protein affinity purification.

An initial phase of library construction (3 weeks) can take place in the home lab under our supervision via an on-line shared labbook. In the second phase, the scientist visits EMBL Grenoble (3 weeks) to participate in automated library screening. The output is a set of clones that have been demonstrated to express solubly and that are purifiable on NiNTA resin.

Funding for transnational access (TNA) is available via the EU PCUBE project and involves an application to PCUBE. The cost of consumables and accommodation during the Grenoble visit will be covered.


Publications

Angelini, A., Tosi, T., Mas, P., Acajjaoui, S., Zanotti, G., Terradot, L. & Hart, D.J. Expression of Helicobacter pylori CagA domains by library-based construct screening. FEBS J. 2009 Feb;276(3):816-24. PubMed

Bonneau, F., Lenherr, E.D., Pena, V., Hart, D.J. & Scheffzek, K. Solubility survey of fragments of the neurofibromatosis type 1 protein neurofibromin. Protein Expr Purif. 2008 Dec 10. PubMed

Tarendeau, F., Crepin, T., Guilligay, D., Ruigrok, R.W., Cusack, S. & Hart, D.J. Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit. PLoS Pathog. 2008 Aug 29;4(8):e1000136. PubMed

Guilligay, D., Tarendeau, F., Resa-Infante, P., Coloma, R., Crepin, T., Sehr, P., Lewis, J., Ruigrok, R.W., Ortin, J., Hart, D.J. & Cusack, S. The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat Struct Mol Biol. 2008 May;15(5):500-6. Epub 2008 May 4. PubMed

Tarendeau, F., Boudet, J., Guilligay, D., Mas, P.J., Bougault, C.M., Boulo, S., Baudin, F., Ruigrok, R.W., Daigle, N., Ellenberg, J., Cusack, S., Simorre, J.P. & Hart, D.J. Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit. Nat Struct Mol Biol. 2007 Mar;14(3):229-33. Epub 2007 Feb 25. PubMed

Alzari, P.M., Berglund, H., Berrow, N.S., Blagova, E., Busso, D., Cambillau, C., Campanacci, V., Christodoulou, E., Eiler, S., Fogg, M.J., Folkers, G., Geerlof, A., Hart, D., Haouz, A., Herman, M.D., Macieira, S., Nordlund, P., Perrakis, A., Quevillon-Cheruel, S., Tarandeau, F., van Tilbeurgh, H., Unger, T., Luna-Vargas, M.P., Velarde, M., Wilmanns, M. & Owens, R.J. Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1103-13. Epub 2006Sep 19. PubMed

Hart, D.J. & Tarendeau, F. Combinatorial library approaches for improving soluble protein expression in Escherichia coli. Acta Crystallogr D Biol Crystallogr. 2006 Jan;62(Pt 1):19-26. Epub 2005Dec 14. PubMed

High-throughput screening for soluble protein expression

In the context of the Partnership for Structural Biology (PSB), combinatorial approaches, screening and robotics are applied to develop novel methods to solve challenging problems of protein expression for structural biology.