Improved cloning and expression of cytochrome P450s and cytochrome P450 reductase in yeast.
Combination of the pYeDP60 yeast expression system with a modified version of the improved uracil-excision (USER) cloning technique provides a new powerful tool for high-throughput expression of eukaryotic cytochrome P450s. The vector presented is designed to obtain an optimal 5' untranslated sequence region for yeast (Kozak consensus sequence), and has been tested to produce active P450s and NADPH-cytochrome P450 oxidoreductase (CPR) after 5' end silent codon optimization of the cDNA sequences. Expression of two plant cytochrome P450s, Sorghum bicolor CYP79A1 and CYP71E1, and S. bicolor CPR2 using the modified pYeDP60 vector in all three cases produced high amounts of active protein. High-throughput functional expression of cytochrome P450s have long been a troublesome task due to the workload involved in cloning of each individual P450 into a suitable expression vector. The redesigned yeast P450 expression vector (pYeDP60u) offers major improvements in cloning efficiency, speed, fidelity, and simplicity. The modified version of the USER cloning system provides great potential for further development of other yeast vectors, transforming these into powerful high-throughput expression vectors.
Hamann
Thomas
T
Plant Biochemistry Laboratory, Department of Plant Biology and Center for Molecular Plant Physiology (PlaCe), Faculty of Life Science, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.
Møller
Birger Lindberg
BL
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D005822:Genetic Vectors; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D045868:Sorghum / Q000201:enzymology