Purification and reactivation of recombinant Synechococcus phytoene desaturase from an overexpressing strain of Escherichia coli.
The Synechococcus phytoene desaturase has been isolated from an overexpressing strain of Escherichia coli. The plasma pPDSde135 mediated the overexpression of the full-length polypeptide directly. The recombinant protein comprised 5% of the total cellular protein and was found predominantly in the inclusion body fraction. Urea was used to solubilize the recombinant protein from the inclusion fraction and the protein was subsequently purified to homogeneity on a DEAE-cellulose column. The purification scheme yielded 4.0 mg of homogeneous desaturase protein after a 20-fold purification, recovering 40% of the original protein from a 100 ml suspension culture of E. coli. The recombinant desaturase had an apparent molecular mass of 53 kDa on SDS/PAGE and crossreacted with an antiserum raised against the expressed protein. Desaturase activity was restored upon the removal of urea. The enzyme catalysed the conversion of phytoene to zeta-carotene via phytofluene. These products of the desaturase reaction existed predominantly in a cis configuration. Lipid replenishment enhanced activity. NAD+ and NADP+ were observed to be involved, whilst FAD was an ineffective electron acceptor.
Fraser
P D
PD
Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, Germany.
Linden
H
H
Sandmann
G
G
D015153:Blotting, Western; D002338:Carotenoids / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004793:Enzyme Reactivators; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014508:Urea / Q000494:pharmacology
D004793:Enzyme Reactivators; D011994:Recombinant Proteins; D002338:Carotenoids; C100185:(all-E) phytoene; D014508:Urea; D010088:Oxidoreductases; C022398:phytoene dehydrogenase