Expression of foreign proteins in chloroplasts: a novel approach using D1-protein fusions

Dirk Fischer and Udo Johanningmeier

Martin-Luther-Universität Halle-Wittenberg, Plant Physiology Institute, Weinbergweg 10, 06120 Halle (Saale), Germany

The D1 protein as a central component of the PSII complex is encoded by the psbA gene located on the chloroplast genome. It is synthesized as a precursor protein with a C-terminal extension, which is cleaved off by the processing protease CtpA and released into the thylakoid lumen. The extension sequence is very diverse among the different organisms and its absence does not significantly affect the function of the protein. In Chlamydomonas reinhardtii the D1 precursor has a telopeptide extension of 8 amino acid residues. We have started to analyse the possibility of a D1-mediated expression of foreign proteins/peptides translationally fused to the extension sequence. This approach should allow us to express light regulated "cut-to-size" polypeptides in plastids. Moreover, since the D1 protein has a fast turnover in the light and is therefore synthesized in high amounts, it should be possible to accumulate stable proteins/peptides by controlling light intensities. Using psbA deletion mutants, transgenic photosynthetic algae can be obtained by selection for photoautotrophic growth. Since no resistance genes are involved, this represents an antibiotic free transgenic approach. As a proof of concept we have successfully expressed a foreign gene by this innovative approach.

e-mail address of presenting author: EMAIL