Optimisation of expression and immobilized metal ion affinity chromatographic purification of recombinant (His)6-tagged cytochrome P450 hydroperoxide lyase in Escherichia coli.
Fatty acid hydroperoxide lyase (HPL) is a cytochrome P450 acting on fatty acid's hydroperoxides in many plants. The optimisation of the expression of recombinant (His)(6)-tagged HPL in Escherichia coli is described: the highest HPL production yield were obtained with TB medium supplemented with 2.5 mM delta-aminolevulinic acid and 0.5 mM IPTG. For the first time, the time course expression of a plant P450 in a bench-scale fermentor is detailed and the amount of recombinant HPL production is 16.3 mg/l. The UV-Visible spectrum of the recombinant (His)(6)-tagged HPL have been recorded after a Ni(2+)-based affinity chromatography (IMAC).
Delcarte
Jérôme
J
Agricultural Research Centre, Chaussee de Namur 146, 5030, Gembloux, Belgium. delcarte@cragx.fgov.be
Fauconnier
Marie- Laure
M
Jacques
Philippe
P
Matsui
Kenji
K
Thonart
Philippe
P
Marlier
Michel
M
D000446:Aldehyde-Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005285:Fermentation; D006639:Histidine / Q000737:chemistry*; D008670:Metals; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet
D008670:Metals; D006639:Histidine; D003577:Cytochrome P-450 Enzyme System; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase