Redirection to thylakoid lumen allows accumulation of recombinant hEGF in transplastomic tobacco

MAURO M. MORGENFELD; E. FEDERICO ALFANO; NOELIA BOCCARDO; FEDERICO MIRKIN; FERNANDO F. BRAVO-ALMONACID

Foz de Iguazú

Congreso; 11th INTERNATIONAL CONGRESS OF PLANT MOLECULAR BIOLOGY; 2015

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins as human epidermal growth factor (hEGF) resulted hindered by post-transcriptional mechanisms. hEGF degradation has been related to redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulphide bonds formation stabilising the functional conformation of the protein. For evaluation of the hypothesis, transplastomic tobacco plants were generated targeting hEGF protein to thylakoid lumen by additioning the transit peptide of the 23 kDa protein of the oxygen-evolving system of photosystem II (Str). This bipartite signal has been previously used to translocate recombinant proteins coded in the nuclear genome into the thylakoid lumen of Arabidopsis thaliana plants.Following this approach hEGF production into the thylakoid lumen reached detectable levels by Western blot exceeding stromal hEGF accumulation that remained undetectable. Southern blot analysis confirms homologous recombination at the plastome and allows discarding different site insertion as explanation of the expression levels. Northern blot analysis supports the upper stability of the hEGF peptide into the thylakoid lumen as primary cause of accumulation increase. Our results highlight the relevance of exploring different strategies to improve recombinant protein stability for certain transgenes in order to exploit potential advantages of recombinant protein accumulation in chloroplasts.