Protéine de fusion

Résumé

Une protéine de fusion est une protéine artificielle obtenue par la combinaison de différentes protéines, ou partie de protéines. Elle est obtenue à la suite de la création par recombinaison de l'ADN d'un gène comportant les cadres de lecture ouverts correspondant aux protéines ou parties de protéines désirées. Les protéines de fusion peuvent également être appelées protéines chimères. Exemples d'utilisation Localisation des protéines Une des applications les plus connues des protéines de fusion est la fusion d'une protéine d'intérêt à une protéine fluorescente. De telles protéines chimères sont utilisées en recherche fondamentale pour étudier la localisation des protéines, c'est-à-dire le positionnement dans la cellule de ces protéines. Purification des protéines purification des protéines Une des étapes importantes de la purification des protéines est la séparation de la protéine d'intérêt des protéines contaminantes. Pour cela, la protéine d'in

Source officielle

https://fr.wikipedia.org/wiki/Prot%C3%A9ine_de_fusion

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Two strategies to improve transient gene expression in HEK293E and/or CHO-DG44 cells

Guillaume Lüthi

Transient gene expression (TGE) is based on episomal DNA expression. This technique is used for the production of recombinant proteins that can be used as therapeutics. TGE is for example extremely useful for shortening the time between candidate proteins screening and their production. Understanding and optimizing TGE is a major concern in the field of recombinant proteins. Thus, we selected here two different approaches to further improve TGE. First, we studied effect of signal peptide (SP) modification on TNFR-Fc protein in transiently transfected HEK293E and CHO-DG44 cells. Secondly, we studied effect of Valproic acid on the production of TNFR-Fc protein in HEK293E cells and on its influence on plasmid methylation. Modification of TNFR-Fc was done with eighteen others SPs. Artificial SPs exhibiting different scores using a prediction method and SPs coming from naturally secreted proteins were tested. None of them could induce a better secretion than the native SP. Nevertheless, they all showed comparable secretion abilities, except one that could not induce protein secretion. We showed here that SP modification could not increase protein secretion in HEK293E or in CHO-DG44. But also that without a valid SP, protein secretion is inhibited. Addition of VPA in HEK293E cells transfected with pXLG-A3 plasmid increased IgG productivity by 11 fold. Nevertheless, we showed here that VPA did not increase TNFR-Fc productivity to the same extent (1.2 fold) even by optimization of its concentration. Furthermore, we investigated the effect of plasmid methylation (TNFRFc and IgG) on TGE using VPA. We found that IgG expression was less influenced by this modification than TNFR-Fc. In fact, TNFR-Fc plasmid methylation drastically decreased specific productivity (0.2 fold). And VPA could almost entirely compensate this effect (0.8 fold). We therefore hypothesized that VPA has an impact on pDNA methylation in the context of TGE.

2011

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Labeling of fusion proteins with small molecular in vivo

Thomas Gronemeyer, Kai Johnsson, Alexandre Juillerat, Antje Keppler

A general method for covalent labeling of fusion proteins in vivo that complements the existing methods for protein labeling in vivo and in vitro is presented. This new method might lead to new ways of studying and manipulating proteins in living cells. Covalent attachment of the small mol. to the fusion protein is achieved by the unusual mechanism of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase, which irreversibly transfers the alkyl group from its substrate, O6-alkylguanine-DNA, to one of its cysteine residues. The applicability of the method in different organisms and its independence of the nature of the label should make this method an important tool for functional studies of proteins in the living cell. [on SciFinder (R)]

Wiley2004

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The authors report on a method for the multicolor imaging of cell surface proteins which is based on the labeling of carrier protein (CP) fusion proteins with different fluorophores. In one application, different generations of a cell surface protein can be sequentially labeled to discriminate between old and newly made copies. In another application, fusions to different CPs can be selectively labeled with different fluorophores in one sample. Both applications open up new ways for studying the properties of cell surface proteins of living cells. [on SciFinder (R)]

2005

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Two strategies to improve transient gene expression in HEK293E and/or CHO-DG44 cells

Guillaume Lüthi

2011