FN Archimer Export Format
PT J
TI MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms
BT 
AF GROISILLIER, Agnes
   HERVE, Christiane
   JEUDY, Alexandra
   REBUFFET, Etienne
   PLUCHON, Pierre Francois
   CHEVOLOT, Yann
   FLAMENT, Didier
   GESLIN, Claire
   MORGADO, Isabel M.
   POWER, Deborah
   BRANNO, Margherita
   MOREAU, Herve
   MICHEL, Gurvan
   BOYEN, Catherine
   CZJZEK, Mirjam
AS 1:1,2;2:1,2;3:1,2;4:1,2;5:3;6:4;7:3;8:3;9:5;10:5;11:6;12:7;13:1,2;14:1,2;15:1,2;
FF 1:;2:PDG-DOP-DCN-SGCN;3:;4:;5:;6:;7:PDG-DOP-DCB-EEP-LMEE;8:;9:;10:;11:;12:;13:;14:;15:;
C1 UPMC Univ Paris 6, LIA DIAMS, UMR Vegetaux Marins & Biomol 7139, Biol Stn, F-29682 Roscoff, France.
   CNRS, LIA DIAMS, UMR Vegetaux Marins & Biomol 7139, Biol Stn, F-29682 Roscoff, France.
   IFREMER, CNRS, UBO, UMR6197, F-29280 Plouzane, France.
   Ecole Cent Lyon, Inst Nanotechnol Lyon INL, UMR5270, F-69134 Ecully, France.
   Univ Algarve, Ctr Ciencias Mar, P-8005139 Faro, Portugal.
   Stn Zool A Dohrn, I-80121 Naples, Italy.
   UPMC, CNRS, Observ Banyuls Sur Mer, UMR7628, Banyuls Sur Mer, France.
C2 UNIV PARIS 06, FRANCE
   CNRS, FRANCE
   IFREMER, FRANCE
   ECOLE CENT LYON, FRANCE
   UNIV ALGARVE, PORTUGAL
   STN ZOOL ANTON DOHRN, ITALY
   UNIV PARIS 06, FRANCE
SI NANTES
   BREST
SE PDG-DOP-DCN-SGCN
   PDG-DOP-DCB-EEP-LMEE
IN WOS Ifremer jusqu'en 2018
   copubli-france
   copubli-europe
   copubli-univ-france
IF 4.544
TC 43
UR https://archimer.ifremer.fr/doc/00011/12219/9003.pdf
LA English
DT Article
AB Background: The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms. Results: We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented. Conclusions: The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.
PY 2010
PD JUL
SO Microbial Cell Factories
SN 1475-2859
PU Biomed Central Ltd
VL 9
UT 000280173600001
DI 10.1186/1475-2859-9-45
ID 12219
ER

EF