Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.1/207
Título: MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms
Autor: Groisillier, Agnes
Herve, Cecile
Jeudy, Alexandra
Rebuffet, Etienne
Pluchon, Pierre F.
Chevolot, Yann
Flament, Didier
Geslin, Claire
Morgado, Isabel
Power, Deborah
Branno, Margherita
Moreau, Herve
Michel, Gurvan
Boyen, Catherine
Czjzek, Mirjam
Data: 14-Jun-2010
Citação: Microbial Cell Factories. 2010 Jun 14;9(1):45
Resumo: Abstract 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.
URI: http://hdl.handle.net/10400.1/207
DOI: http://dx.doi.org/10.1186/1475-2859-9-45
Aparece nas colecções:CCM2-Artigos (em revistas ou actas indexadas)

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