Heterologous production of oligopeptides with antihypertensive effects in Lactuca sativa and Medicago truncatula

Abstract

“In the current scenario of a worldwide problematic situation related to cardiovascular diseases associated with hypertension, the necessity for alternative therapies has become a great challenge. The need for therapies with little side effects and with the same efficacy, but less expensive, has given the opportunity to the new biotechnological paradigm brought by molecular pharming to win its market share from the established pharmaceutical industry. Molecular pharming is a costeffective, scalable and safe system to produce high-quality and biologically active recombinant therapeutic proteins. Medicago truncatula and Lactuca sativa (lettuce) are two emerging plant hosts for molecular pharming. M. truncatula is a model legume plant amenable for transformation and in vitro manipulation, along with the procedural extrapolation to other legume species. L. sativa is a worldwide raw edible plant which allows the oral delivery of recombinant therapeutic products. In the present work, the heterologous production of four ACEI peptides - that have proven antihypertensive effect – in Medicago and lettuce hosts was analyzed. The ACEI peptides production was verified at leaf level in the two plant production hosts. An in vitro plant stock was kept to allow the progression of the work, and a seed bank was established to allow further studies in the progeny. The presence of the ACEI coding sequences transcripts in Medicago RNA samples confirmed the stable transformation of this host. In the lettuce host, an in vitro seed selection scheme based on kanamycin was established to screen ACEI transformants which were further analyzed at genomic DNA level confirming the stable plant transformation with ACEI coding sequences. At protein production level, protocols for the peptide extraction, along with ELISA, SDS-PAGE, western immunoblotting, and HPLC were established for further detection and quantification of ACEI peptide production.”