Modifications in the N-terminus of an insect cytochrome P450 enhance production of catalytically active protein in baculovirus-Sf9 cell expression systems.
Although baculovirus vectors are powerful tools for the heterologous expression of proteins in insect cell cultures, some insect and plant microsomal P450 proteins are not effectively expressed in this system. Hypothesizing that their expression failures might result from collisions between their N-terminal sequences and adjacent cytosolic sequences, we compared and mutated the N-terminus of Papilio multicaudatus CYP6B33, which is inappropriately folded in Sf9 cells, to sequences present in its Papilio polyxenes CYP6B1 counterpart, which is efficiently expressed and appropriately folded. Molecular modeling of the three differences in the linker separating the signal anchor domain (SAD) and the cytosolic domain identified Val32 in CYP6B33 as a residue potentially important for folding and/or positioning of the cytosolic domain. Mutation of Val32 to Ala32 in the CYP6B33 linker (CYP6B33 V32A mutant) or replacement of the CYP6B33 SAD with that of CYP6B1 (CYP6B1 1-20/CYP6B33 21-500 mutant) allowed for significant P450 expression, indicating that complex interactions involving both the signal anchor and membrane linker affect folding and activity of P450s in this heterologous expression system.
Mao
Wenfu
W
Department of Entomology, University of Illinois, Urbana, IL 61801, USA.
Berenbaum
May R
MR
Schuler
Mary A
MA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016367:Baculoviridae; D002080:Butterflies / Q000201:enzymology*; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015870:Gene Expression; D008730:Methoxsalen / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D008730:Methoxsalen