Abstract
DP1B is a synthetic analogue of spider dragline silk protein. It can be spun to form silk fiber. Previously, it had been expressed in transgenic plants, showing the general feasibility of the plant-based DP1B production. However, success of such a plant-based platform requires a great increase of DP1B productivity in plant cells to reduce production cost. This report describes a protein targeting approach to accumulate DP1B in apoplast, ER lumen, and vacuole in Arabidopsis cells, by utilizing appropriate combinations of sporamin-targeting determinant peptides and ER retention peptide. The approach has dramatically enhanced DP1B accumulation, resulting in high production yield. The accumulation can be as high as 8.5 and 6.7% total soluble protein in leaf tissue by targeting to apoplast and ER lumen, respectively, or as high as 18 and 8.2% total soluble protein in seeds by targeting to ER lumen and vacuole, respectively. However, the vacuole targeting in leaves and the apoplast targeting in seeds have failed to accumulate full length DP1B molecules or any DP1B at all, respectively, suggesting that they may not be suitable for applications in leaf tissues and seeds. Data in this study recommend a combination of seed-specific expression and ER-targeting as one of the best strategies for yield enhancement of plant-based DP1B production.
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References
S Arcidiacono C Mello D Kaplan S Cheley H Bayley (1998) ArticleTitlePurification and characterization of recombinant spider silk expressed in Escherichia coli Appl Microbiol Biotechnol 49 31–38 Occurrence Handle9487707
LA Barr SR Fahnestock J Yang (2004) ArticleTitleProduction and purification of recombinant DP1B silk-like protein in plants Mol Breeding 13 345–356
N Bechtold J Elli G Pelletier (1993) ArticleTitleIn planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants C R Acad Sci Paris, Life Sci 316 1194–1199
P Boevink B Martin K Oparka SS Cruz C Hawes (1999) ArticleTitleTransport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A Planta 208 392–400
PG Caimi LM McCole TM Klein PS Kerr (1996) ArticleTitleFructan accumulation and sucrose metabolism in transgenic maize endosperm expressing a Bacillus amyloliquefaciens SacB gene Plant Physiol 110 355–363 Occurrence Handle12226187
J Cappello JW Crissman (1990) ArticleTitleThe design and production of bioactive protein polymers for biomedical application Polymer Preprints 31 193–194
J Cappello J Crissman M Dorman M Mikolajczak G Textor M Marquet F Ferrari (1990) ArticleTitleGenetic engineering of structural protein polymers Biotechnol Prog 6 198–202 Occurrence Handle1366613
MJ Chrispeels (1991) ArticleTitleSorting of proteins in the secretory system Annu Rev Plant Physiol Plant Mol Biol 42 21–53
U Conrad U Fiedler (1998) ArticleTitleCompartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity Plant Mol Biol 38 101–109 Occurrence Handle9738962
SR Fahnestock LA Bedzyk (1997) ArticleTitleProduction of synthetic spider dragline silk protein in Pichia pastoris Appl Microbiol Biotechnol 47 33–39 Occurrence Handle9035408
SR Fahnestock SL Irwin (1997) ArticleTitleSynthetic spider dragline silk proteins and their production in Escherichia coli Appl Microbiol Biotechnol 47 23–32 Occurrence Handle9035407
SR Fahnestock Z Yao LA bedzyk (2000) ArticleTitleMicrobial production of spider silk protein J Biotechnol 74 105–119 Occurrence Handle11763501
T Hattori T Nakagawa M Maeshima K Nakamura T Asahi (1985) ArticleTitleMolecular cloning and nucleotide sequence of cDNA for sporamin, the major soluble protein of sweet potato tuberous roots Plant Mol Biol 5 313–320
EM Herman BA Larkins (1999) ArticleTitleProtein storage bodies and vacuoles Plant Cell 11 601–613 Occurrence Handle10213781
DL Kaplan CM Mello S Arcidiacono S Fossey K Senecal W Muller (1997) Silk K McGrath D Kaplan (Eds) Protein-Based Material Birkhauser Boston, USA 104–131
A Lazaris S Arcidiacono Y Huang J-F Zhou F Duguay N Chretien EA Welsh JW Soares CN Karatzas (2002) ArticleTitleSpider silk fibers spun from soluble recombinant silk produced in mammalian cells Science 295 472–476 Occurrence Handle11799236
RV Lewis M Hinman S Kothakota MJ Fournier (1996) ArticleTitleExpression and purification of a spider silk protein: a new strategy for producing repetitive proteins Protein Expr Purif 7 400–406 Occurrence Handle8776759
Liu Z-B and Odell JT 1999. Specific gene activation by chimeric Gal4 transcription factors in stable transgenic plants. US patent No: US5968793.
M Maeshima T Sasaki T Asahi (1985) ArticleTitleCharacterization of major proteins in sweet potato tuberous roots Phytochemistry 24 1899–1902
K Matsuoka S Matsumoto T Hattori Y Machida K Nakamura (1990) ArticleTitleVacuolar targeting and posttranslational processing of the precursor to the sweet potato tuberous root storage protein in heterologous plant cells J Biol Chem 265 19750–19757 Occurrence Handle2246259
K Matsuoka K Nakamura (1991) ArticleTitlePropeptide of a precursor to a plant vacuolar protein required for vacuolar targeting Proc Natl Acad Sci USA 88 834–838 Occurrence Handle1992474
R Menassa H Zhu C Karatzas A Lazaris A Richman J Brandle (2004) ArticleTitleSpider dragline silk proteins in transgenic tobacco leaves: accumulation and field production Plant Biotech J 2 431–438
MM Moloney LA Holbrook (1997) ArticleTitleSubcellular targeting and purification of recombinant proteins in plant production systems Biotechnol Genet Eng Rev 14 321–336 Occurrence Handle9188158
EE Murray J Lotzer M Eberle (1989) ArticleTitleCodon usage in plant genes Nucleic Acids Res 17 477–498 Occurrence Handle2644621
JT Prince KP McGrath CM DiGirolamo DL Kaplan (1995) ArticleTitleConstruction, cloning, and expression of synthetic genes encoding spider dragline silk Biochemistry 34 10879–10885 Occurrence Handle7662669
J Scheller K-H Guhrs F Grosse U Conrad (2001) ArticleTitleProduction of spider silk proteins in tobacco and potato Nature Biotechnol 19 573–577
J Scheller D Henggeller A Viviani U Conra (2004) ArticleTitlePurification of spider-elastin from transgenic plants and application for human chondrocyte proliferation Transgenic Res 13 51–57 Occurrence Handle15070075
PR Shewry JA Napier AS Tatham (1995) ArticleTitleSeed storage proteins: structures and biosynthesis Plant Cell 7 945–956 Occurrence Handle7640527
J Yang GC Fox SuffixJr TV Henry-Smith (2003) ArticleTitleIntein-mediated assembly of a functional β-glucuronidase in transgenic plants Proc Natl Acad Sci USA 100 3513–3518 Occurrence Handle12629210
MT Ziegler SR Thomas KJ Danna (2000) ArticleTitleAccumulation of a thermostable endo-1,4-β-D-glucanase in the apoplast of Arabidopsis thaliana leaves Mol Breeding 6 37–46
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Yang, J., Barr, L.A., Fahnestock, S.R. et al. High yield recombinant silk-like protein production in transgenic plants through protein targeting. Transgenic Res 14, 313–324 (2005). https://doi.org/10.1007/s11248-005-0272-5
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DOI: https://doi.org/10.1007/s11248-005-0272-5