Subcloning Cytochrome P450 Mutants to Increase Mutant Protein Expression Yield

Our laboratory is developing hybrid P450 enzymes capable of the selective oxidation of various substrate C-H bonds upon light activation. The proposed hybrid enzymes contain a photosensitizer covalently attached to single non-native cysteine mutants of the P450 BM3 heme domain. A library of non-native single cysteine mutants has been generated using site directed mutagenesis on the pC-Wori expression vector encoding the P450BM3 heme domain gene. However, differences in mutant protein expression have been observed to date, hampering further development of the hybrid enzymes. In order to increase the overall protein expression yield in all mutants, we are working on a subcloning method. We hypothesize that subcloning will increase mutant protein expression yield. The subcloning method consists of cutting the mutant gene from one vector using BamHI and EcoRI restriction enzymes and inserting it into a high yield expression vector using ligase. Our goal is to produce single non-native cysteine P450 BM3 mutants with consistently high protein expression yield. As proof of concept, several low expression mutant coding regions have been inserted into the high expression vector and expressed. The results from comparative studies between the original and the current method will be presented.