GENERATION OF THE CYTOSOLIC DOMAIN OF MICROSOMAL P450 52A3 AFTER HIGH-LEVEL EXPRESSION IN SACCHAROMYCES CEREVISIAE

Publisher Summary This chapter provides a method to produce the cytosolic domain of P450Cm1 in a scale and quality suitable for further studies on structure-function relationships. Only very low amounts of the cytosolic domain of P450Cm1 are available after its direct biosynthesis in Saccharomyces cerevisiae . Possible reasons include instability, which is rapid degradation of the truncated protein, and impaired folding in the absence of proper membrane integration could be discussed in analogy to recent reports on other P450 forms. The method discussed in the chapter may be useful if the otherwise easier cDNA deletion approach yields only poor results. The expression levels and properties of the modified P450 have to be analyzed in comparison to that of the wild-type protein. Each of the different microsomal P450 forms requires some specific conditions to avoid denaturation and, at the same time, improve the efficiency of the cleavage reaction.