A single cupredoxin azurin production in transplastomic tobacco

Abstract

The single-copper protein azurin from Pseudomonas aeruginosa has attracted great interest as an anti-cancer therapeutic agent or as a fuel cell catalyst for energy conversion. In this work, we obtained transgenic tobacco plants transformed with the chloroplast expression vector harboring the mature azurin polypeptide fused to psbA 5′UTR element, confirmed the integration of site-specificity into the tobacco chloroplast genome through homologous recombination by Southern hybridization analysis, and also identified the maternal inheritance. Northern hybridization analysis showed the polycistronic transcription expression pattern of the azurin gene. In addition, post-transcriptional processing of azurin monocistron was observed, which may be due to the endonucleolytic and intercistronic cleavage of the psbA mRNA 5′UTR element. Also, we examined the azurin expression levels depending on leaf maturity, showing a high expression level of 5.7 % of total soluble protein (TSP) in young leaves, in contrast to a low expression level of 0.72 % TSP in fully mature leaves. In addition, the copper level of transplastomic chloroplasts increased by twofold compared with that of non-transplastomic chloroplasts. These results suggest that the increased copper level may be due to the production of azurin in transplastomic chloroplasts, representing the formation of active azurin with copper ions in active sites.