On-column refolding of an insoluble histidine tag recombinant exopolyphosphatase from Trypanosoma brucei overexpressed in Escherichia coli.
An exopolyphosphatase gene has been cloned by polymerase chain reaction (PCR) from Trypanosoma brucei and the corresponding protein overexpressed as a recombinant His-tag (histidine tag) exopolyphosphatase in E. coli in order to characterize its biochemical activity and to produce antibody to determine its localization. Because overexpression of this protein in bacteria resulted in the formation of inactive inclusion bodies, these structures were first solubilized in denaturant condition (6 M urea). Secondly, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column from 6 M to 0 M urea in the presence of 1% Triton X-100. Triton X-100 was used to abolish protein aggregation during the refolding step. The purified enzyme was active, demonstrating that at least part of the proteins was properly refolded.
Lemercier
G
G
Laboratoire de Parasitologie Moléculaire, Université Victor Segalen Bordeaux 2, 146 Rue Léo Saignat, 33076 cedex, Bordeaux, France.
Bakalara
N
N
Santarelli
X
X
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006639:Histidine / Q000737:chemistry*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D017931:DNA Primers; D011994:Recombinant Proteins; D006639:Histidine; D017766:Acid Anhydride Hydrolases; C020137:exopolyphosphatase