Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

Abstract

SummaryWe have engineered the chloroplast of eukaryotic algae to produce a number of recombinant proteins, including human monoclonal antibodies, but, to date, have achieved expression to only 0.5% of total protein. Here, we show that, by engineering the mammalian coding region of bovine mammary-associated serum amyloid (M-SAA) as a direct replacement for the chloroplast psbA coding region, we can achieve expression of recombinant protein above 5% of total protein. Chloroplast-expressed M-SAA accumulates predominantly as a soluble protein, contains the correct amino terminal sequence and has little or no post-translational modification. M-SAA is found in mammalian colostrum and stimulates the production of mucin in the gut, acting in the prophylaxis of bacterial and viral infections. Chloroplast-expressed and purified M-SAA is able to stimulate mucin production in human gut epithelial cell lines. As Chlamydomonas reinhardtii is an edible alga, production of therapeutic proteins in this organism offers the potential for oral delivery of gut-active proteins, such as M-SAA.

A Chlamydomonas zöldalga nemzetség algáinak szerepe a biotechnológiában és helyük a zöldalgák rendszerében

“…A gyógyászati alkalmazás lehetőségei A Chlamydomonas reinhardtii tenyészetekre "fehérje-bioreaktor"-ként is tekinthetünk, ugyanis az általuk termelt rekombináns fehérjék gyógyászati célokat szolgálhatnak (Manuel et al 2007). A rekombináns DNS-technológia kidolgozása megteremtette az alapjait annak, hogy a terápiás célra használandó fehérjé-ket egyszerű mikroorganizmusok által, a fermentáció jól ismert alapelveit alkalmazva termeltessék (Mayfield és Franklin 2005).…”

A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (HGAD65)

“…Indeed, the C. reinhardtii chloroplast expression of a large single-chain antibody has shown accumulation levels of 0.5 to 1% of algal TSP [22]. Recently, Manuell et al [23] demonstrated robust expression of a bioactive mammalian peptide, bovine mammary-associated serum amyloid (M-SAA), in C. reinhardtii chloroplasts with levels up to 5% of TSP. There are several other antigenic proteins that have been produced using this system, including foot-and-mouth disease virus VP1 protein [24], tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) [25] and the protein E2 of classical swine fever virus [26].…”

The microalgaChlamydomonas reinhardtiias a platform for the production of human protein therapeutics

“…This is consistent with previous published data. 14,15 Indirect immunofluorescence of transgenic C. reinhardtii stably expressing HMGB1-1xFLAG verifies the chloroplast localization of the biopharmaceutical as well as demonstrates the large volume that the single algal chloroplast occupies within the cell ( fig. 2).…”

“…Many of the proteins that have been quantified have been shown to accumulate as high as 2-20% of total soluble protein (TSP). [14][15][16] Importantly, the chloroplast contains the proper machinery to form disulfide bonds and assemble chloroplast. 16 VEGF and HMGB1 were purified from algal lysates and tested for bioactivity.…”

“…6 C. reinhardtii grows relatively quickly, doubling every 5-8 hours, and can grow to densities above 10 7 cells/ml. Because C. reinhardtii is photosynthetic, its media pD1-KanR except that the recombinant gene is cloned downstream of the well expressed M-SAA gene, 15 resulting in a M-SAA fusion protein, and (3) pAtpA in which expression of the human therapeutic gene is controlled by the atpA promoter and 5' UTR and the rbcL 3' UTR and integrates into an intergenic region of the chloroplast genome near the psbA locus ( fig. 1).…”