A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera

Madesis, Panagiotis; Osathanunkul, M.; Georgopoulou, Urania; Gisby, Martin F.; Mudd, Elisabeth A.; Nianiou, I.; Tsitoura, Panagiota; Mavromara, Penelope; Tsaftaris, Athanasios; Day, Anil

doi:10.1016/j.jbiotec.2009.12.001

Cited by 27 publications

(36 citation statements)

References 64 publications

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“…Thus, increased protein production in these strains highlights the necessity for codon optimization of any gene for which high levels of protein production are desired. Further, in a recent study, a hepatitis C virus core polypeptide expressed in chloroplasts, the results suggested that the codonoptimised gene increased monocistronic core mRNA levels by at least 2-fold and core polypeptides by over 5-fold, relative to the native viral gene [35]. …”

Section: Factors For High-yield Production In Chloroplast Expressimentioning

confidence: 99%

Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

Gao

Xue

et al. 2012

Journal of Biomedicine and Biotechnology

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Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed.

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Section: Factors For High-yield Production In Chloroplast Expressimentioning

confidence: 99%

Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

Gao

Xue

et al. 2012

Journal of Biomedicine and Biotechnology

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“…In vitro assays of inserted genes with several synonymous codons show that translation efficiency does not always correlate with codon usage in plastid mRNAs (Nakamura and Sugiura, 2007), but they have been used in several codon optimization studies (Lutz et al, 2001;Ye et al, 2001;Franklin et al, 2002;Lenzi et al, 2008;Jabeen at al., 2010;Madesis et al, 2010;Gisby et al, 2011;Wang et al, 2015b;Boehm et al, 2016;Nakamura et al, 2016). While some studies achieved significant increases in expression (75-to 80-fold) after codon optimization (Franklin et al, 2002;Gisby et al, 2011), other studies observed negligible enhancement (Ye et al, 2001;Lenzi et al, 2008;Daniell et al, 2009;Wang et al, 2015b;Nakamura et al, 2016).…”

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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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“…Methods for DNA-and RNA-blot preparation and hybridization have been described (Madesis et al, 2010). RNA molecular standards were from New England Biolabs.…”

Section: Nucleic Acid-blot Analysesmentioning

confidence: 99%

“…IPTG (1 mM) induction was for 2 h once bacterial cells reached an optical density at 600 nm of 0.4 (BioPhotometer Spectrophotometer; Eppendorf). Sedimented pET30-dao induced and uninduced cells were concentrated 20-fold, relative to the original culture volume, in sample buffer (62.5 mM TrisHCl, pH 6.8, 10% [v/v] (Madesis et al, 2010). For DAAO enzyme assays, induced pET30b-dao and control (pET30b empty vector) E. coli strains were concentrated 10-fold in cell extract buffer (0.1 M Tris, pH 8, 1 mM EDTA, 0.15 M KCl, 5% glycerol, and 0.6% Triton X-100), lysed by sonication, and sedimented (5,000 rpm; Sorvall SS34 rotor), and the supernatant was used for assays.…”

Section: Nucleic Acid-blot Analysesmentioning

confidence: 99%

Growth of Transplastomic Cells Expressing d-Amino Acid Oxidase in Chloroplasts Is Tolerant to d-Alanine and Inhibited by d-Valine

2012

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Dual-conditional positive/negative selection markers are versatile genetic tools for manipulating genomes. Plastid genomes are relatively small and conserved DNA molecules that can be manipulated precisely by homologous recombination. Highyield expression of recombinant products and maternal inheritance of plastid-encoded traits make plastids attractive sites for modification. Here, we describe the cloning and expression of a dao gene encoding D-amino acid oxidase from Schizosaccharomyces pombe in tobacco (Nicotiana tabacum) plastids. The results provide genetic evidence for the uptake of D-amino acids into plastids, which contain a target that is inhibited by D-alanine. Importantly, this nonantibiotic-based selection system allows the use of cheap and widely available D-amino acids, which are relatively nontoxic to animals and microbes, to either select against (D-valine) or for (D-alanine) cells containing transgenic plastids. Positive/negative selection with D-amino acids was effective in vitro and against transplastomic seedlings grown in soil. The dual functionality of dao is highly suited to the polyploid plastid compartment, where it can be used to provide tolerance against potential D-alanine-based herbicides, control the timing of recombination events such as marker excision, influence the segregation of transgenic plastid genomes, identify loci affecting dao function in mutant screens, and develop D-valine-based methods to manage the spread of transgenic plastids tagged with dao.

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A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera

Cited by 27 publications

References 64 publications

Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

Growth of Transplastomic Cells Expressing d-Amino Acid Oxidase in Chloroplasts Is Tolerant to d-Alanine and Inhibited by d-Valine

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