Copper amine oxidases (CAO) are a group of enzymes that catalyze the conversion of primary amines to aldehydes and ammonium, reducing O2 to H2O2 in the process, and utilizing 2,4,5-trihydroxyphenylalanyl quinone (TPQ) and Cu2+ as cofactors. Research suggests that CAOs may use different mechanisms depending on the specific homolog. In order to gain a better understanding of why different homologs proceed through different mechanisms, more homologs must be studied. The aim of this research is to develop an expression system in plants for various homologs of CAOs. Initially we will target hVAP1 (a human CAO), BSAO (bovine serum amine oxidase), and possibly PSAO (a plant CAO). Plant expression systems have been developed for commercial production of some proteins, including hirudin and ZMapp. Expression of green fluorescent protein (GFP) has also been achieved in Nicotiana benthamiana plants through the use of Agrobacterium tumefaciens infiltration. This research seeks to utilize this system to express different homologs of CAOs in plants. The first step in this project is to express GFP in N. benthamiana as a proof of principle. To do this, a plant expression vector with the gene for GFP needed to be constructed. Site directed mutagenesis was performed on specific areas of GFP’s genetic sequence so that it could properly anneal into the vector. The next step in this research will be to transform this vector into A. tumefaciens, and then introduce the Agrobacterium into the N. benthamiana plants through vacuum infiltration. The expression of GFP by the plant will be evaluated using a UV light, since GFP fluoresces. If successful, this process will be repeated for the different CAO homologs. Expression of CAOs in plants will allow us to study the mechanisms of these enzymes.

Plant expression system for copper amine oxidases

251st National Meeting (2016)

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