Enhanced expression of CYP1B1 in Escherichia coli

Conditions for the optimal expression of the human CYP1B1 hemoprotein in Es cherichia coli have been investigated. CYP1B1 cDNA was prepared From a reti nal cDNA template and used to generate cDNA fragments with modified 5'-sequ ences reported to allow enhanced expression in E. coli DH5 alpha. Plasmids were constructed, using the pCWori + expression vector and were used to exa mine necessity for thiamine. F-aminolevulinic acid (ALA), and IPTG. The opt imal shaking speed in an orbital incubator was 150 rpm at 30 degrees C. Hig her speeds resulted in increased cell death and lower speeds resulted in lo wer expression of cytochrome P450. IPTG was necessary for this expression s ystem, which makes use of the lac repressor, but levels above 0.5 mM were w ithout additional benefit. We were able to show thiamine to be unnecessary in this expression system: although included by others expressing CYP1B1. A LA has been reported to enhance expression of several different forms of cy tochrome P450. We examined the dependence of CYP1B1 expression on ALA. The expression proved to be highly dependent upon this heme precursor, with lev els of CYP1B1 increasing similar to 20-fold, to 920 nmol/l in the presence of up to 2.5 mM ALA. The question of whether heme synthesis and apoprotein synthesis were coupled was then investigated. It could be shown that althou gh heme synthesis was not limiting (CYP101 holoenzyme expression in the abs ence of ALA was four times higher than the ALA-supported CYP1B1 holoenzyme expression), it was necessary for optimal expression of CYP1B1. CYP1B1 prot ein synthesis appears to be coupled to heme precursor availability, as seen by SDS-PAGE, because in the absence of heme precursor apocytochrome P450 1 B1 does not accumulate. (C) 2000 Elsevier Science Ireland Ltd. All rights r eserved.