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MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

2010-06-14Journal article Open access
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dc.contributor.authorGroisillier, Agnes
dc.contributor.authorHerve, Cecile
dc.contributor.authorJeudy, Alexandra
dc.contributor.authorRebuffet, Etienne
dc.contributor.authorPluchon, Pierre F.
dc.contributor.authorChevolot, Yann
dc.contributor.authorFlament, Didier
dc.contributor.authorGeslin, Claire
dc.contributor.authorMorgado, Isabel
dc.contributor.authorPower, Deborah
dc.contributor.authorBranno, Margherita
dc.contributor.authorMoreau, Herve
dc.contributor.authorMichel, Gurvan
dc.contributor.authorBoyen, Catherine
dc.contributor.authorCzjzek, Mirjam
dc.date.accessioned2011-06-03T13:53:29Z
dc.date.available2011-06-03T13:53:29Z
dc.date.issued2010-06-14
dc.date.updated2011-06-03T13:53:29Z
dc.description.abstractAbstract 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.
dc.description.versioninfo:eu-repo/semantics/publishedVersion
dc.identifier.citationMicrobial Cell Factories. 2010 Jun 14;9(1):45
dc.identifier.doihttp://dx.doi.org/10.1186/1475-2859-9-45
dc.identifier.otherAUT: DPO00386;
dc.identifier.urihttp://hdl.handle.net/10400.1/207
dc.language.isoeng
dc.rights.holderBiomedCentral
dc.titleMARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms
dc.typejournal article
dspace.entity.typePublication
person.familyNameMorgado
person.familyNamePower
person.givenNameIsabel
person.givenNameDeborah Mary
person.identifier.ciencia-id3315-7715-C3B4
person.identifier.ciencia-id891A-8A44-3CAE
person.identifier.orcid0000-0002-7826-997X
person.identifier.orcid0000-0003-1366-0246
person.identifier.ridJ-4798-2013
person.identifier.scopus-author-id18134268300
person.identifier.scopus-author-id7101806760
rcaap.rightsopenAccess
rcaap.typearticle
relation.isAuthorOfPublication67026aa2-ccdb-4b68-9938-b09e0fd6aca0
relation.isAuthorOfPublicationc68f5ffb-63f6-4c70-8957-29e464fb59c0
relation.isAuthorOfPublication.latestForDiscoveryc68f5ffb-63f6-4c70-8957-29e464fb59c0

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