Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field production.

Spider dragline silk is a unique biomaterial and represents nature's strongest known fibre. As it is almost as strong as many commercial synthetic fibres, it is suitable for use in many industrial and medical applications. The prerequisite for such a widespread use is the cost-effective production in sufficient quantities for commercial fibre manufacturing. Agricultural biotechnology and the production of recombinant dragline silk proteins in transgenic plants offer the potential for low-cost, large-scale production. The purpose of this work was to examine the feasibility of producing the two protein components of dragline silk (MaSp1 and MaSp2) from Nephila clavipes in transgenic tobacco. Two different promoters, the enhanced CaMV 35S promoter (Kay et al., 1987) and a new tobacco cryptic constitutive promoter, tCUP (Foster et al., 1999) were used, in conjunction with a plant secretory signal (PR1b), a translational enhancer (alfalfa mosaic virus, AMV) and an endoplasmic reticulum (ER) retention signal (KDEL), to express the MaSp1 and MaSp2 genes in the leaves of transgenic plants. Both genes expressed successfully and recombinant protein accumulated in transgenic plants grown in both greenhouse and field trials.

[1]  M. Van Montagu,et al.  Gene silencing results in instability of antibody production in transgenic plants , 1999, Molecular and General Genetics MGG.

[2]  Ashutosh Chilkoti,et al.  Purification of recombinant proteins by fusion with thermally-responsive polypeptides , 1999, Nature Biotechnology.

[3]  T. Ouellet,et al.  A tobacco cryptic constitutive promoter, tCUP, revealed by T-DNA tagging , 1999, Plant Molecular Biology.

[4]  Anthony Acampora,et al.  Last mile by laser. , 2002, Scientific American.

[5]  Annie Bellemare,et al.  High‐toughness Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells , 2003 .

[6]  M. Sirard,et al.  Making recombinant proteins in animals--different systems, different applications. , 2003, Trends in biotechnology.

[7]  Fritz Vollrath,et al.  Liquid crystalline spinning of spider silk , 2001, Nature.

[8]  J. Macaulay.  Biopharming: Growing Medicine Crops Science, Communications, and Government Relations , 2003 .

[9]  Steven Arcidiacono,et al.  Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells , 2002, Science.

[10]  Lee Gehrke,et al.  Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence , 1987, Nature.

[11]  J. Scheller,et al.  Production of spider silk proteins in tobacco and potato , 2001, Nature Biotechnology.

[12]  P. Zambryski,et al.  The transfer of DNA from agrobacterium tumefaciens into plants: a feast of fundamental insights. , 2000, The Plant journal : for cell and molecular biology.

[13]  J. Bol,et al.  Circadian expression and induction by wounding of tobacco genes for cysteine proteinase , 1993, Plant Molecular Biology.

[14]  J. Brandle,et al.  Transgenic Tobacco: Gene Expression and Applications , 1999 .

[15]  W. Stiekema,et al.  The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco , 1996, Plant Molecular Biology.

[16]  L. Bedzyk,et al.  Production of synthetic spider dragline silk protein in Pichia pastoris , 1997, Applied Microbiology and Biotechnology.

[17]  M B Hinman,et al.  Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber. , 1992, The Journal of biological chemistry.

[18]  D. Klessig,et al.  Isolation and nucleotide sequence of cDNA clones for the pathogenesis-related proteins PR1a, PR1b and PR1c of Nicotiana tabacum cv. Xanthi nc induced by TMV infection. , 1988, Nucleic acids research.

[19]  Paul Christou,et al.  Genetic modification: The production of recombinant pharmaceutical proteins in plants , 2003, Nature Reviews Genetics.

[20]  M B Hinman,et al.  Synthetic spider silk: a modular fiber. , 2000, Trends in biotechnology.

[21]  M. Montagu,et al.  An Agrobacterium-mediated transient gene expression system for intact leaves , 1997 .

[22]  J. Howard,et al.  Production of recombinant proteins in transgenic plants: Practical considerations , 1997, Biotechnology and bioengineering.

[23]  K. Wu,et al.  A constitutive gene expression system derived from the tCUP cryptic promoter elements , 2002, Theoretical and Applied Genetics.

[24]  Robert Kay,et al.  Duplication of CaMV 35S Promoter Sequences Creates a Strong Enhancer for Plant Genes , 1987, Science.

[25]  A. Jevnikar,et al.  A self-contained system for the field production of plant recombinant interleukin-10 , 2001, Molecular Breeding.

[26]  S. Fahnestock,et al.  Synthetic spider dragline silk proteins and their production in Escherichia coli , 1997, Applied Microbiology and Biotechnology.

[27]  R. Lewis,et al.  Structure of a protein superfiber: spider dragline silk. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[28]  D. Kaplan,et al.  Construction, cloning, and expression of synthetic genes encoding spider dragline silk. , 1995, Biochemistry.