title: Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation.
abstract: 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 codon-optimized 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.
authors.affiliation: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6030 (K.-C.K., H.-T.C., H.D.);Global Research, Novo Nordisk, Malov DK-2760, Denmark (I.R.L.); andInstitute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229 (R.W.-C., A.B.).
authors.forename: Kwang-Chul
authors.identifier: 0000-0002-4037-1776
authors.initials: KC
authors.lastname: Kwon
authors.affiliation: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6030 (K.-C.K., H.-T.C., H.D.);Global Research, Novo Nordisk, Malov DK-2760, Denmark (I.R.L.); andInstitute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229 (R.W.-C., A.B.).
authors.forename: Hui-Ting
authors.initials: HT
authors.lastname: Chan
authors.affiliation: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6030 (K.-C.K., H.-T.C., H.D.);Global Research, Novo Nordisk, Malov DK-2760, Denmark (I.R.L.); andInstitute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229 (R.W.-C., A.B.).
authors.forename: Ileana R
authors.identifier: 0000-0001-7319-2080
authors.initials: IR
authors.lastname: León
authors.affiliation: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6030 (K.-C.K., H.-T.C., H.D.);Global Research, Novo Nordisk, Malov DK-2760, Denmark (I.R.L.); andInstitute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229 (R.W.-C., A.B.).
authors.forename: Rosalind
authors.initials: R
authors.lastname: Williams-Carrier
authors.affiliation: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6030 (K.-C.K., H.-T.C., H.D.);Global Research, Novo Nordisk, Malov DK-2760, Denmark (I.R.L.); andInstitute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229 (R.W.-C., A.B.).
authors.forename: Alice
authors.initials: A
authors.lastname: Barkan
authors.affiliation: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6030 (K.-C.K., H.-T.C., H.D.);Global Research, Novo Nordisk, Malov DK-2760, Denmark (I.R.L.); andInstitute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229 (R.W.-C., A.B.) hdaniell@upenn.edu.
authors.forename: Henry
authors.identifier: 0000-0003-4485-1176
authors.initials: H
authors.lastname: Daniell
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism; D003062:Codon / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D018545:Lactuca / Q000235:genetics / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D014176:Protein Biosynthesis / Q000235:genetics*; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D019076:Transgenes / Q000235:genetics
chemical_list: D003062:Codon; D045332:Photosystem II Protein Complex; D020543:Proteome