Séminaire

Séminaire de l’IPBS
Renaud Vincentelli
UMR 7257, CNRS – Aix-Marseille université. Architecture et Fonction des
Macromolécules Biologiques (AFMB), Marseille
Mercredi 11 Mai 2016 à 11h
Salle des séminaires IPBS
High Throughput methods for protein production, protein-DNA
and protein-protein interaction studies.
Escherichia coli is the most widely used expression system for the production of recombinant proteins
for structural and functional studies. However, purifying proteins is sometimes challenging since many
proteins are expressed in an insoluble form without optimization. It is therefore recommended to use
high throughput protein expression screening on a small scale (1-4 ml cultures) to identify the successful
conditions for production. To this end, together with the help of recent and HTP flexible cloning
strategies, custom made protocols that enabled the systematic screening of culture conditions and
fusion-tags at a pace of more than a thousand cultures per week (1, 2, 3) have been developed in the lab.
The protocols have been validated on thousands of proteins for customers and collaborators at AFMB.
The statistics generated allowed the selection of the simplest protocol that gives the maximum soluble
expression yield for the production of proteins upon scale up, and in the microgram range at the
screening scale. This procedure has been validated, for instance, with the production of the majority of
the DNA binding domains of Ciona intestinalis (1) and the production of the full human PDZ domain
repertoire (4). In 2015, with a modification of this procedure (5) the team have produced and purified
few thousands recombinant disulfide rich toxins for the European FP7 VENOMICS project. To
characterize proteins at a pace and scale that is compatible with the microgram range yields, custom
made protocols have been developed in the lab like a new in vitro protein- DNA interaction assay (HTP
SELEX (6)), a HTP Pull-down and a new HTP and quantitative in vitro protein-protein interaction
assays (HTP Hold-up (4)).
1) High-throughput protein expression screening and purification in Escherichia coli. Vincentelli R, Cimino A,
Geerlof A, Kubo A, Satou Y, Cambillau C. Methods. 2011 Sep;55 (1):65-72
2) High-Throughput Expression Screening and Purification of Recombinant Proteins in E. coli. N. J. Saez, R.
Vincentelli, Methods Mol Biol. 2014;1091:33-53
3) High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom
proteins produced in E. coli. Saez, NJ, Nozach H, Blemont, M, Vincentelli, R. JOVE, 2014 Jul 30;(89):e51464.
4) Quantifying domain-ligand affinities and specificities by high-throughput holdup assay. *Vincentelli R, Luck K*,
Poirson J, Polanowska J, Abdat J, Blémont M, Turchetto J, Iv F, Ricquier K, Straub ML, Forster A, Cassonnet P,
Borg JP, Jacob Y, Masson M, Nominé Y, Reboul J, Wolff N, Charbonnier S, Travé G. Nat Methods. 2015
Aug;12(8):787-93
5) High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant
expression of small disulfide-rich proteins in E. coli. Nozach N*, Fruchart-Gaillard C*, Fenaille F, Beau F,
Henrique Pereira Ramos O, Douzi B, Saez N. J, Moutiez M, Servent D, Gondry M, Thaï R, Cuniasse P,
Vincentelli R* and Dive V*, equal contributors, Microbial Cell Factories 2013, 12:37
6) DNA-binding specificities of human transcription factors. Jolma A, Yan J, Whitington T, Toivonen J, Nitta KR,
Rastas P, Morgunova E, Enge M, Taipale M, Wei G, Palin K, Vaquerizas JM, Vincentelli R, Luscombe NM,
Hughes TR, Lemaire P, Ukkonen E, Kivioja T, Taipale J., Cell. 2013 Jan 17;152 (1-2):327-39.
Contact: [email protected], [email protected]
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