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FEBS Lett. 1995 Nov 6;374(3):345-50.

Cinnamate 4-hydroxylase from Catharanthus roseus, and a strategy for the functional expression of plant cytochrome P450 proteins as translational fusions with P450 reductase in Escherichia coli.

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1
Institut für Biologie II, Universität Freiburg, Germany.

Abstract

A PCR-based approach was used to isolate cDNAs for cinnamate 4-hydroxylase (C4H) from Catharanthus roseus cell cultures. The protein shared 75.9% identity with C4H from other plants, and the transcription was induced under various stress conditions. The cloned protein was used to investigate the functional expression of plant P450/P450-reductase fusions in E. coli. Fusions containing a modified N-terminal membrane anchor were located in the membrane and possessed C4H activity without solubilization or addition of other factors. The results indicate that the fusion protein strategy provides a useful tool to analyze the activities encoded in the rapidly increasing number of plant P450 sequences of uncertain or unknown function. We also discuss critical elements of the strategy: the choice of the E. coli host strain, the N-terminal membrane anchor, and the conditions for protein expression.

PMID:
7589568
[Indexed for MEDLINE]
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