Chromatographic purification of an insoluble histidine tag recombinant Ykt6p SNARE from Arabidopsis thaliana over-expressed in E. coli.
In order to undertake in plant cell the study of the endoplasmic reticulum (ER)-Golgi apparatus (GA) protein and/or lipid vesicular transport pathway, expressed sequence tag (EST) coding for a homologue to the yeast soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Ykt6p has been cloned in Arabidopsis thaliana by reverse transcription polymerase chain reaction (RT-PCR). The corresponding protein was over-expressed as a recombinant histidine-tag (his-tag) protein in E. coli. Starting from one litter of culture, an ultrasonic homogenization was performed for cell disruption and after centrifugation the Arabidopsis Ykt6p SNARE present in inclusion bodies in the pellet was solubilized. After centrifugation, the clarified feedstock obtained was injected onto an immobilized metal affinity chromatography (IMAC) in presence of 6 M guanidine and on-column refolding was performed. Folded and subsequently purified (94% purity) recombinant protein was obtained with 82% of recovery.
Vincent
Patrick
P
Laboratoire de Biogenèse Membranaire, CNRS-UMR 5544, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.
Dieryck
Wilfrid
W
Maneta-Peyret
Lilly
L
Moreau
Patrick
P
Cassagne
Claude
C
Santarelli
Xavier
X
D017360:Arabidopsis / Q000737:chemistry*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D006639:Histidine