title: Spider silk-like proteins derived from transgenic Nicotiana tabacum.
abstract: The high tensile strength and biocompatibility of spider dragline silk makes it a desirable material in many engineering and tissue regeneration applications. Here, we present the feasibility to produce recombinant proteins in transgenic tobacco Nicotiana tabacum with sequences representing spider silk protein building blocks . Recombinant mini-spidroins contain native N- and C-terminal domains of major ampullate spidroin 1 (rMaSp1) or rMaSp2 flanking an abbreviated number (8, 16 or 32) of consensus repeat domains. Two different expression plasmid vectors were tested and a downstream chitin binding domain and self-cleavable intein were included to facilitate protein purification. We confirmed gene insertion and RNA transcription by PCR and reverse-transcriptase PCR, respectively. Mini-spidroin production was detected by N-terminus specific antibodies. Purification of mini-spidroins was performed through chitin affinity chromatography and subsequent intein activation with reducing reagent. Mini-spidroins, when dialyzed and freeze-dried, formed viscous gelatin-like fluids.
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, 130 McGinty Court, 153 Robert F. Poole Agricultural Center, Clemson, SC, 29634, USA.
authors.forename: Congyue Annie
authors.initials: CA
authors.lastname: Peng
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, 130 McGinty Court, 153 Robert F. Poole Agricultural Center, Clemson, SC, 29634, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: Russo
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, 130 McGinty Court, 153 Robert F. Poole Agricultural Center, Clemson, SC, 29634, USA.|College of Pharmacy, Medical University of South Carolina, Charleston, SC, 29425, USA.
authors.forename: Charlene
authors.initials: C
authors.lastname: Gravgaard
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, 130 McGinty Court, 153 Robert F. Poole Agricultural Center, Clemson, SC, 29634, USA.|Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, 37232, USA.
authors.forename: Heather
authors.initials: H
authors.lastname: McCartney
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, 130 McGinty Court, 153 Robert F. Poole Agricultural Center, Clemson, SC, 29634, USA.
authors.forename: William
authors.initials: W
authors.lastname: Gaines
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, 130 McGinty Court, 153 Robert F. Poole Agricultural Center, Clemson, SC, 29634, USA. marcotw@clemson.edu.
authors.forename: William R
authors.initials: WR
authors.lastname: Marcotte
mesh_terms: D002846:Chromatography, Affinity; D005349:Fibroins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005612:Freeze Drying; D047668:Inteins / Q000235:genetics; D030821:Plants, Genetically Modified; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; C086087:spidroin 1; C076815:spidroin 2; D005349:Fibroins
keywords: Affinity chromatography; Intein; Spider silk; Spidroin; Transgenic tobacco