Factors effecting expression of vaccines in microalgae

Surzycki, Raymond; Greenham, Kathleen; Kitayama, Kaoru; Dibal, Flora; Wagner, Richard; Rochaix, Jean David; Ajam, Tarek; Surzycki, Stefan

doi:10.1016/j.biologicals.2009.02.005

Cited by 167 publications

(126 citation statements)

References 18 publications

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“…There is a drawback of using a codon table based on all chloroplast genes, which assumes that all tRNA species are equally abundant. However, such translational selection is not possible (Surzycki et al, 2009). Therefore, in this study, we developed a codon optimizer program based on the codon usage of psbA genes across 133 plant species to increase the expression of heterologous genes in chloroplasts.…”

Section: Discussionmentioning

confidence: 99%

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

et al. 2016

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Codon optimization based on psbA genes from 133 plant species eliminated 105 (human clotting factor VIII heavy chain [FVIII HC]) and 59 (polio VIRAL CAPSID PROTEIN1 [VP1]) rare codons; replacement with only the most highly preferred codons decreased transgene expression (77-to 111-fold) when compared with the codon usage hierarchy of the psbA genes. Targeted proteomic quantification by parallel reaction monitoring analysis showed 4.9-to 7.1-fold or 22.5-to 28.1-fold increase in FVIII or VP1 codon-optimized genes when normalized with stable isotope-labeled standard peptides (or housekeeping protein peptides), but quantitation using western blots showed 6.3-to 8-fold or 91-to 125-fold increase of transgene expression from the same batch of materials, due to limitations in quantitative protein transfer, denaturation, solubility, or stability. Parallel reaction monitoring, to our knowledge validated here for the first time for in planta quantitation of biopharmaceuticals, is especially useful for insoluble or multimeric proteins required for oral drug delivery. Northern blots confirmed that the increase of codonoptimized protein synthesis is at the translational level rather than any impact on transcript abundance. Ribosome footprints did not increase proportionately with VP1 translation or even decreased after FVIII codon optimization but is useful in diagnosing additional rate-limiting steps. A major ribosome pause at CTC leucine codons in the native gene of FVIII HC was eliminated upon codon optimization. Ribosome stalls observed at clusters of serine codons in the codon-optimized VP1 gene provide an opportunity for further optimization. In addition to increasing our understanding of chloroplast translation, these new tools should help to advance this concept toward human clinical studies.

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Section: Discussionmentioning

confidence: 99%

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

et al. 2016

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“…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).…”

mentioning

confidence: 88%

“…8 It has been shown in both algae and in higher plants that recombinant proteins accumulate to much higher levels when expressed from the chloroplast genome in comparison to the nuclear genome. 9,10 The chloroplast of C. reinhardtii has been shown to support expression of a range of recombinant proteins including reporters such as GUS, 11 luciferase, 12,13 and GFP, 9 vaccines, 14 and industrial enzymes (our unpublished data). Many of the proteins that have been quantified have been shown to accumulate as high as 2-20% of total soluble protein (TSP).…”

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confidence: 89%

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

2011

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“…As is the case for most heterologous genes, optimizing the codon usage of algae-destined transgenes to reflect this bias increases their expression efficiency by increasing their translation rates and may decrease their susceptibility to silencing (Heitzer et al, 2007, Potvin and. In prokaryotic or prokaryotic-derived genomes, such as chloroplasts from eukaryotic algae, codon bias is one of the most critical elements for protein expression (Surzycki et al, 2009). The importance of codon optimization in marine algal genetic transformation applications is increasingly acknowledged in recent transgenic research (Lerche andHallmann, 2009, Takahashi et al, 2010).…”

Section: Vector Construction: Promoter Selection and Codon Usagementioning

confidence: 99%