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Etude de l'ATPase Ca2+ du réticulum sarco/endoplasmique : Mise au point d'une nouvelle méthode de purification de SERCA1a de lapin exprimée chez S. cerevisiae permettant sa cristallisation et applications au mutant E309Q-Etude d'une autre isoforme, SERCA3a

Abstract : We describe here a new purification method of the rabbit SERCA1a after its heterologous expression in the yeast S. cerevisiae . A “biotin acceptor domain” is fused at the C-terminal part of the protein and is biotinylated in vivo by the yeast. The purification procedure, based on the strong interaction between avidin and biotin, allows us to obtain a protein pure at 40-50% and well active. After an additional purification step by SEC-HPLC the protein is pure at about 70% and always well active. Crystals were obtained from SERCA1a purified by this method and they diffract X-rays at 3,1 Å.
This new method was used with sucicess to purify the mutant E309Q. Small crystals of the mutant were obtained. SERCA3a could also be purified using this procedure, but the low yield of expression limits the amount that could be purified. In parallel, immunolocalization of SERCA3a was performed in several cell lines and in cryosections of skin.
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https://tel.archives-ouvertes.fr/tel-00011204
Contributor : Marie Habets Jidenko <>
Submitted on : Friday, June 23, 2006 - 3:50:57 PM
Last modification on : Friday, October 23, 2020 - 4:51:51 PM

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  • HAL Id : tel-00011204, version 2

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Marie Habets Jidenko. Etude de l'ATPase Ca2+ du réticulum sarco/endoplasmique : Mise au point d'une nouvelle méthode de purification de SERCA1a de lapin exprimée chez S. cerevisiae permettant sa cristallisation et applications au mutant E309Q-Etude d'une autre isoforme, SERCA3a. Biochimie [q-bio.BM]. Université Paris Sud - Paris XI, 2005. Français. ⟨tel-00011204v2⟩

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