Therapeutic recombinant proteins have become major players in the treatment and prevention of disease and are currently in high demand. To date, various recombinant expression systems have been used in a variety of model systems, with bacteria being the most widely used. Recently, microalgae have gained interest as potential bioreactors for recombinant protein production and offer important advantages of both prokaryotic and eukaryotic expression systems. The single-celled green algae Chamydomonas reihardtii is a favoured recombinant protein production host due to many beneficial characteristics including advanced genome informatics for both the nucleus and organelles, efficient and effective nuclear plastid transformation systems, rapid transformant screening, and capacity for both phototrophic and heterotrophic growth. However, to date no industrial-scale applications have been established due to inefficient expression and low target protein yield. The 3’ polyadenylate (poly(A)) tail on mRNA is a post transcriptional modification that impacts mRNA expression and protein translation by stabilising and facilitating the transport of mRNA out of the nucleus in addition to increasing gene expression efficiency. The length of the tail increases the stability and half life of mRNA thereby increasing the chance of translation. Canonical polyadenylate polymerases (cPAPSs) are genes that encode poly(A) polymerases which are involved in the synthesis of the poly(A) tail at the 3’ end of the mRNA transcript. C. reihardtii have only one copy of the cPAPS gene, therefore these researchers sought to overexpress the cPAPS gene to determine if overexpression of the poly(A) polymerase could result in increased protein translation and accumulation. Canonical C. reihardtii poly (A) polymerase (crePAPSs) overexpression lines were generated and confirmed by semi-quantitative PCR and quantitative PCR (qPCR). After expressing crePAPS polyadenylate in Escherichia coli and purifying the protein by Ni-NTA resin, the polyadenylate activity of crePAPS was confirmed by polyadenylation assays. The results revealed that CrePAPS did exhibit polyadenylate activity and increases in protein concentration resulted in a longer poly(A) tail. Subsequent label-free quantitation (LFQ) analysis confirmed overexpression of CrePAPS resulted in an overall increase in protein accumulation when compared to wild type lines. Additionally, an increase in crude and dissolved protein content was confirmed by Kjeldahl and bicinchoninic acid (BCA) assay, verifying an increase in translation in crePAPS overexpressing lines. Further analysis showed a total of 1447 upregulated differentially accumulated proteins (DAPs) and 94 proportionally altered proteins (PAPs) in the overexpressing lines; 48 DAPs and 2 PAPs were upregulated in all overexpressing strains. GO and KEGG enrichment analysis of the upregulated DAPs and PAPs showed the greatest enrichment categories and pathways were associated with ribosomal assembly, offering further support to an increase translation in the overexpressing lines. Subcellular location analysis of upregulated DAPs and PAPs revealed cytoplasmic, chloroplast, and mitochondrial proteins showed the greatest increase which may provide a reference for the selection of recombinant proteins. Taken together, these findings provide insight into the ability to exploit C. reihardtii for recombinant protein production on an industrial scale.
How was PEAKS used?
Raw MS/MS data files from the Orbitrap Fusion Eclipse were analysed using PEAKS Xpro. Protein identification was performed by searching the data against a UniProt database with a precursor mass tolerance of 10 ppm, a fragment ion mass tolerance of 0.02 Da, and a maximum of 3 missed cleavages was allowed. Carbamidomethylation [C] was set as a fixed post-translational modification (PTM) and oxidation [M] and deamidation [NQ] were selected as modified PTMs. Confident protein identification required a minimum of one unique peptide, and proteins were filtered using an 1% FDR using a target-decoy strategy. For protein quantification, two unique peptides were required, ion intensity changes were used, and the quantitative protein ratios were weighted and normalised against the median ratio.
Wang, Quan, et al. “Overexpressing CrePAPS Polyadenylate Activity Enhances Protein Translation and Accumulation in Chlamydomonas reinhardtii.” Marine Drugs 20.5 (2022): 276. https://doi.org/10.3390/md20050276
The alga Chlamydomonas reinhardtii is a potential platform for recombinant protein expression in the future due to various advantages. Dozens of C. reinhardtii strains producing genetically engineered recombinant therapeutic protein have been reported. However, owing to extremely low protein expression efficiency, none have been applied for industrial purposes. Improving protein expression efficiency at the molecular level is, therefore, a priority. The 3′-end poly(A) tail of mRNAs is strongly correlated with mRNA transcription and protein translation efficiency. In this study, we identified a canonical C. reinhardtii poly(A) polymerase (CrePAPS), verified its polyadenylate activity, generated a series of overexpressing transformants, and performed proteomic analysis. Proteomic results demonstrated that overexpressing CrePAPS promoted ribosomal assembly and enhanced protein accumulation. The accelerated translation was further verified by increased crude and dissolved protein content detected by Kjeldahl and bicinchoninic acid (BCA) assay approaches. The findings provide a novel direction in which to exploit photosynthetic green algae as a recombinant protein expression platform.