Factors effecting expression of vaccines in microalgae Raymond Surzycki a , Katie Greenham a , Kaoru Kitayama a , Flora Dibal a , Richard Wagner a, * , Jean-David Rochaix b , Tarek Ajam c , Stefan Surzycki c a PhycoBiologics Inc., Indianapolis IN, 3416 Ashwood Drive, Bloomington, IN 47401, USA b University of Geneva, Geneva, Switzerland c Department of Biology, Indiana University, Bloomington, USA Abstract PhycoBiologics is developing an oral vaccine delivery system using vaccines expressed in the chloroplast of microalgae. Despite many advances in plastid transformation technology, levels of expression remain inconsistent. We have concluded that the main factors affecting the level of recombinant protein expression in the chloroplast of Chlamydomonas are: codon optimization, protease activity, protein toxicity and transformation-associated genotypic modification. Ó 2009 Published by Elsevier Ltd on behalf of The International Association for Biologicals. Keywords: Chlamydomonas; Recombinant protein expression; Chloroplast transformation 1. Introduction Despite many advances in plastid transformation tech- nology, protein expression levels have remained inconsistent in our and other’s laboratories. Efforts reported in the litera- ture, and subsequently applied in our laboratory to increase protein expression have focused primarily on the roles of promoters, 5 0 and 3 0 UTR sequences, construction of tran- scriptional fusions between native and recombinant peptides, and codon optimization. However, a comparison of the scientific literature to our own data reveals that this often leads to contradictory results. We transformed the chloroplast of Chlamydomonas with over 20 recombinant proteins (10 vaccines), within several chloroplast loci and analyzed the levels of protein expression. This approach has permitted us to comprehensively evaluate the factors affecting the level of protein expression in these algae. Our result indicate that the main factors affecting recombinant protein expression levels are correct codon optimization, protein toxicity, protease activity and transformation-associated genotypic modification, the transformosome. 2. Material and methods 2.1. Strains and growth conditions Chlamydomonas reinhardtii strains CC741 mt(þ) and Fud7 mt() were obtained from the Chlamydomonas Culture Collection. Both strains contain a psbA gene deletion. Strains were maintained on TAP medium either in constant light (70e100 mEm 2 s 1 ) or in the dark at temperature of 24  C. Transformants were grown under the same conditions. 2.2. Cloning and transformation Codon optimization of foreign genes was carried out using a standard assembly PCR method. A FLAG epitope was added to the C terminal of each gene. Codon optimized genes were ligated into pBA155 or pSR229 vectors. The pBA155 vector contains (a) the psbA promoter and terminator for driving the expression of the transgene, (b) the aadA gene [1] for selection of transformants and (c) flanking psbA sequences for homol- ogous integration into inverted repeat of cpDNA. The pSR 229 plasmid was derived from the previously described p-228 plasmid [2] obtained from the Chlamydomonas Culture Collections and contains a 16S spr mutations (resistance to * Corresponding author. Tel.: þ1 812 339 3057; fax: þ1 812 333 7396. E-mail address: wagner@phycotransgenics.com (R. Wagner). 1045-1056/09/$36.00 Ó 2009 Published by Elsevier Ltd on behalf of The International Association for Biologicals. doi:10.1016/j.biologicals.2009.02.005 Available online at www.sciencedirect.com Biologicals 37 (2009) 133e138 www.elsevier.com/locate/biologicals