Optimisation of expression and purification of the recombinant Yol066 (Rib2) protein from Saccharomyces cerevisiae.
Yeast protein Yol066 (encoded by YOL066 ORF, also known as Rib2) possesses two distinct sequence domains: C-terminal deaminase domain and N-terminal part related to RNA:pseudouridine (psi)-synthases. The deaminase domain is implicated in the riboflavine biosynthesis, while the exact function of the RNA:Psi-synthase domain remains obscure. Here we report the optimisation of growth conditions and purification scheme for recombinant His(6)-tagged Yol066 expressed in E. coli BL21(DE3) using pET28 plasmid. Production of soluble Yol066 protein is best at low temperature (18 degrees C) and IPTG concentration (50 micro M) and Yol066 purification was achieved using metal-affinity and ion-exchange chromatography. This optimised protocol yields about 10 mg of highly purified recombinant Yol066 from 3 l of E. coli culture.
Urban
A
A
Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP Nancy I, Faculté des Sciences, BP 239, 54506 Cedex, Vandoeuvre-les-Nancy, France.
Ansmant
I
I
Motorin
Y
Y
D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification*
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins