Expression and Purification of a Spider Silk Protein: A New Strategy for Producing Repetitive Proteins

Lewis, Randolph V.; Hinman, Michael; Kothakota, Srinivas; Fournier, Maurille J.

doi:10.1006/prep.1996.0060

Cited by 164 publications

(118 citation statements)

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“…Synthesis of pBI121-SS tob -YK20-EGFP, pBI121-SS tob -YK8-EGFP, and pBI121-SS tob -YL8-EGFP-Gene synthesis of tomato P3 extensin analogs encoding repeats of the consensus sequences SPPPPSPSPPP-PYYYK (YK) and SPPPPSPSPPPPYYYL (YL) was accomplished using a modified version of a method described earlier (31). Briefly, mutually priming sense and antisense oligonucleotide pairs (supplemental Fig.…”

Section: Methodsmentioning

confidence: 99%

Di-isodityrosine Is the Intermolecular Cross-link of Isodityrosine-rich Extensin Analogs Cross-linked in Vitro

Held

Li²,

Kamyab³

et al. 2004

Journal of Biological Chemistry

107

129

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Extensins are cell wall hydroxyproline-rich glycoproteins that form covalent networks putatively involving tyrosyl and lysyl residues in cross-links catalyzed by one or more extensin peroxidases. The precise cross-links remain to be chemically identified both as network components in muro and as enzymic products generated in vitro with native extensin monomers as substrates. However, some extensin monomers contain variations within their putative cross-linking motifs that complicate cross-link identification. Other simpler extensins are recalcitrant to isolation including the ubiquitous P3-type extensin whose major repetitive motif, Ser-(Hyp) 4 -Ser-Hyp-Ser-(Hyp) 4 -Tyr-Tyr-Tyr-Lys, is of particular interest, not least because its Tyr-Tyr-Tyr intramolecular isodityrosine cross-link motifs are also putative candidates for further intermolecular cross-linking to form di-isodityrosine. Therefore, we designed a set of extensin analogs encoding tandem repeats of the P3 motif, including Tyr 3 Phe and Lys 3 Leu variations. Expression of these P3 analogs in Nicotiana tabacum cells yielded glycoproteins with virtually all Pro residues hydroxylated and subsequently arabinosylated and with likely galactosylated Ser residues. This was consistent with earlier analyses of P3 glycopeptides isolated from cell wall digests and the predictions of the Hyp contiguity hypothesis. The tyrosine-rich P3 analogs also contained isodityrosine, formed in vivo. Significantly, these isodityrosine-containing analogs were further crosslinked in vitro by an extensin peroxidase to form the tetra-tyrosine intermolecular cross-link amino acid diisodityrosine. This is the first identification of an intermolecular cross-link amino acid in an extensin module and corroborates earlier suggestions that di-isodityrosine represents one mechanism for cross-linking extensins in muro.Hydroxyproline-rich glycoproteins (HRGPs), 1 which include the extensins, proline-rich proteins, and arabinogalactan proteins (AGPs), contribute to extracellular matrix architecture throughout the plant kingdom and the chlorophycean green algae (1-3). HRGPs are involved in all aspects of plant growth and development, including wall assembly during embryogenesis (4), and responses to biotic and abiotic stress that include mechanical stress (5), physical wounding (6), pathogenesis (7), and symbiosis (8 -10).HRGPs are extended macromolecules consisting of small repetitive peptide and glycopeptide motifs that form peptide modules and glycomodules of functional significance, as in "mix-and-match" mode they define the molecular properties of the overall macromolecule. The glycomodules result from a combination of post-translational modifications unique to plants, namely proline hydroxylation (11) and its subsequent glycosylation (12) that leads either to short arabino-oligosaccharide or larger arabinogalactan polysaccharide addition to the Hyp residues. A sequence-dependent O-Hyp glycosylation code directs the addition of oligosaccharide and polysaccharides (13), and it is likely that...

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Section: Methodsmentioning

confidence: 99%

Di-isodityrosine Is the Intermolecular Cross-link of Isodityrosine-rich Extensin Analogs Cross-linked in Vitro

Held

Li²,

Kamyab³

et al. 2004

Journal of Biological Chemistry

107

129

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“…Gatesy et al, 2001;Guerette et al, 1996;Hayashi and Lewis, 1998;Simmons et al, 1996;Tian and Lewis, 2005). Finally, the mechanical process of spinning fibers from liquid dope has been studied from both morphological and functional perspectives (Garrido et al, 2002;Vollrath and Knight, 1999;Vollrath et al, 1998;Vollrath et al, 2001) and genetic engineers are synthesizing recombinant silk proteins (Arcidiacono et al, 1998;Foo and Kaplan, 2002;Huemmerich et al, 2004;Lazaris et al, 2002;Lewis et al, 1996;Winkler et al, 1999). Together, these disparate fields of research indicate the potential of the spider silk system to integrate aspects of organismal biology, molecular evolution and biomechanics (e.g.…”

Section: Introductionmentioning

confidence: 99%

Silken toolkits: biomechanics of silk fibers spun by the orb web spiderArgiope argentata(Fabricius 1775)

Blackledge

Hayashi

2006

Journal of Experimental Biology

258

275

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SUMMARY Orb-weaving spiders spin five fibrous silks from differentiated glands that contain unique sets of proteins. Despite diverse ecological functions, the mechanical properties of most of these silks are not well characterized. Here,we quantify the mechanical performance of this toolkit of silks for the silver garden spider Argiope argentata. Four silks exhibit viscoelastic behaviour typical of polymers, but differ statistically from each other by up to 250% in performance, giving each silk a distinctive suite of material properties. Major ampullate silk is 50% stronger than other fibers, but also less extensible. Aciniform silk is almost twice as tough as other silks because of high strength and extensibility. Capture spiral silk, coated with aqueous glue, is an order of magnitude stretchier than other silks. Dynamic mechanical properties are qualitatively similar, but quantitatively vary by up to 300% among silks. Storage moduli are initially nearly constant and increase after fiber yield, whereas loss tangents reach maxima of 0.1–0.2 at the yield. The remarkable mechanical diversity of Argiope argentata silks probably results in part from the different molecular structures of fibers and can be related to the specific ecological role of each silk. Our study indicates substantial potential to customize the mechanics of bioengineered silks.

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“…Many different hosts, including bacteria (13,14), yeast (15), baculovirus/insect systems (16)(17)(18), mammalian cells (19), transgenic plants (20), and transgenic animals were used to produce spider silk proteins. These efforts were successful, as each yielded spider silk proteins, but none of these hosts provided high yields; most were expensive to scale up, and none was naturally equipped to spin silk fibers.…”

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confidence: 99%

Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties

Teulé

Miao

Sohn

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

239

158

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The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.biomaterials | biotechnology | transgenic animals

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Expression and Purification of a Spider Silk Protein: A New Strategy for Producing Repetitive Proteins

Cited by 164 publications

References 0 publications

Di-isodityrosine Is the Intermolecular Cross-link of Isodityrosine-rich Extensin Analogs Cross-linked in Vitro

Di-isodityrosine Is the Intermolecular Cross-link of Isodityrosine-rich Extensin Analogs Cross-linked in Vitro

Silken toolkits: biomechanics of silk fibers spun by the orb web spiderArgiope argentata(Fabricius 1775)

Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties

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