Heterologous expression and purification of rat cytochrome P450 1A1

Cytochrome P450 1A1 belongs to "superfamily" of cytochromes P450 (CYPs). It is an extrahepatic enzyme that takes a part in detoxification metabolism of many xenobiotics but unfortunately also it is one of the most important representatives of CYPs involved in the activation of procarcinogens. The main aim of this bachelor thesis was to prepare and purify cytochrome P450 1A1 in sufficient quantity and purity by heterologous expression. Two different vectors, which were constructed by inserting the gene for rat cytochrome P450 1A1 in efficient expression plasmid pCW, were prepared during this bachelor thesis. Functionality of prepared vectors was verified by measuring the expression of CYP1A1 and CO difference spectra. It was found that only one of the vectors mentioned above provides native protein. This vector was transformed into E. coli cells of strain DH5α, DH5α with the inserted plasmid pHg1 and DH5α with inserted plasmid pGRO7. Various conditions of CYP1A1 production were tested: growth media (LB or TB medium), the time of production (24 and 48h), the concentration of IPTG (0.5mM and 1mM) and the presence of ALA. The highest expression level was achieved in E. coli DH5α cells cultivated in a modified TB medium after 48 hours of production and at 30řC (induction at OD600 0.6 to 0.8 by 0.5mM..