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At least 721 records · Page 40Linked to original sources
PubMed2006

Design of superior spider silk: from nanostructure to mechanical properties.

Spider dragline silk is of practical interest because of its excellent mechanical properties. However, the structure of this material is still largely unknown. In this article, we report what we believe is a new model of the hierarchical structure of silk based on scanning electron microscope and atomic force microscope images. This hierarchical structure includes beta-sheet, polypeptide chain network, and silk fibril. It turns out that an exceptionally high strength of the spider dragline silk can be obtained by decreasing the size of the crystalline nodes in the polypeptide chain network while increasing the degree of orientation of the crystalline nodes. Based on this understanding, how the reeling speed affects mechanical properties of spider dragline silk can be understood properly. Hopefully, the understanding obtained in this study will shed light on the formation of spider silk, and consequently, on the principles for the design of ultrastrong silk.

Animals↗
PubMed2004

Phylogenetic placement of the spider genus Nephila (Araneae: Araneoidea) inferred from rRNA and MaSp1 gene sequences.

The family status of the genus Nephila, which belongs to Tetragnathidae currently but Araneidae formerly, was reexamined based on molecular phylogenetic analyses. In the present study, 12S and 18S rRNA gene fragments of eight species of spiders were amplified and sequenced. In addition, 3'-end partial cDNA of major ampullate spidroin-1 (MaSp1) gene of Argiope amoena was cloned and sequenced, and the 3'-end non-repetitive region's cDNA sequence of MaSp1 gene and the predicted amino acid sequence of C-terminal non-repetitive region of MaSp1 were aligned with some previously known sequences. The resulting phylogeny showed that Araneidae and Tetragnathidae are not a sister group in the superfamily Araneoidea, and the genus Nephila is closer to the genera of the family Araneidae rather than to those of Tetragnathidae. We suggest that the genus Nephila should be transferred back to Araneidae. Or the subfamily Nephilinae might be elevated to family level after it was redefined and redelimited. Furthermore, the study showed that 3'-end non-repetitive region's cDNA sequence of MaSp1 gene and C-terminal non-repetitive region's amino acid sequence of MaSp1 are useful molecular markers for phylogenetic analysis of spiders.

Amino Acid Sequence↗
PubMed2002

[Study on construct and expression of synthetic genes encoding spider dragline silk in Escherichia coli].

Dragline spider silk produced from Nephilia clavipes major ampullate is a natural fibrous protein with specific mechanical properties such as high tensile strength and elasticity. Synthetic gene monomer encoding recombinant spider silk protein, based on the known repetitive protein sequence and partial cDNA sequence of dragline silk, was constructed and expressed. DNA monomer sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimer was cloned into pET30a(+), a prokaryotic high potency expression vector, and induced with IPTG. The protein from 8-unit repeat was produced in Escherichia coli at levels up to 20 mg/L. The protein was easily purified with high recovery by using an metal ion affinity chromatography and purity was over 90%. The results of SDS-PAGE and Western blot suggested that the mass of the expression product was about 37 kD. This value and amino acid analysis were consistent with those of theoretic calculation.

Amino Acid Sequence↗
PubMed2002

[Amplification, cloning and sequence analysis of spider dragline silk cDNA].

Spider dragline silk is synthesized in special gland named major ampulate (MA) gland. The MA glands were dissected from the abdomen of the spiders Nephila clavata and the total RNA was extracted by the TRIZOL. The cDNA of dragline silk was amplificated by RT-PCR (reverse transcription polymerase chain reaction), multiplex PCR and cloned. PCR identification, restriction analysis and DNA sequence analysis were carried out to verify the recombinant plasmids. The codon usage frequencies of the cloned cDNA were added up, and the predicted amino acid sequence was compared with Spidroin2 of Nephila clavipes. Predicted secondary structure of the predicted amino-acid sequence was analysized by DNAStar software. All results showed that the cloned cDNA we got (GenBank Accession No. AF441245) was the very fragment of spider dragline silk Spidroin2 cDNA.

Amino Acid Sequence↗
PubMed2003

[Production and application of rabbit anti-imitative spider dragline silk protein polyclonal antibody].

A synthetic spider dragline gene s600 was cloned into fusion protein expression vector pGEX-KG and expressed in Escherichia coli. Protein S600 was purified and rabbit antiserum was prepared. Amino acid composition analysis confirmed the right expression of S600. Western blot analysis revealed that anti-S600 antiserum could react with natural spider silk, so the synthetic dragline protein, designed by the authors, shares similar immunological characteristics with the natural spider silk. An ELISA system was also established for the quantitative detection of synthetic dragline protein expression in silk gland (or in the cocoon) of transgenic silkworm.

Animals↗
PubMedDate not supplied

[Construction of the synthetic genes for protein analogs of spider silk carcass spidroin 1 and their expression in tobacco plants].

To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f9 coding for spidroin 1 analogs were 3'-fused in-frame with the reporter lichenase gene. The Tr2' weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.

Agrobacterium tumefaciens↗
PubMed2004

[Silk protein fiber biomaterials and tissue engineering].

OBJECTIVE: To summarize the latest developments in silk protein fiber as biomaterials and their applications in tissue engineering. METHODS: Recent original literature on silk protein fiber as biomaterials were reviewed, illustrating the properties of silk protein fiber biomaterials. RESULTS: The silk protein fiber has the same functions of supporting the cell adhesion, differentiation and growth as native collagen, and is renewed as novel biomaterials with good biocompatibility, unique mechanical properties and is degradable over a longer time. CONCLUSION: Silk protein fiber can be used as a suitable matrix for three dimensional cell culture in tissue engineering. It has a great potential applications in other fields.

Animals↗
PubMedDate not supplied

[The properties of natural silk fibers: deformation study and NMR data].

Natural silk (Bombyx mori) fibers with low humidity (0.07 g H2O/g dried silk) after temperature influence were studied for mechanical longitudinal deformation. On the basis of the stress-strain curves, some estimates of tensile properties for silk fibers were obtained. It was found that the maximal tension (sigma(max) in tensile-linear field of deformation of silk fibers decreases with increasing fiber diameter. The results showed that the heating of fibers (100 degrees C) results in a diminishing of the sigma(max)-value. Scanning electron microscopy pictures for cross section and longitudinal fiber surface were obtained. Natural silk fibers were studied by the NMR relaxation method (free induction decay curves) and the second moments of NMR-line shape in silk samples were calculated. The intra- and intermolecular contributions into the second moment were analyzed. The results showed a strong interaction of water molecules with macromolecules and a low molecular mobility. Some characteristics of interactions between silk macromolecules and water molecules as well as the role of intermolecular links in the change of the structure-function properties of natural silk under the action of external factors are discussed.

Animals↗
PubMedDate not supplied

[A study of periodicity in the primary structure of spidroin 1 and spidroin 2 from spiders belonging to various species].

Sequences of spidroin 1 and 2 from spiders belonging to various species were analyzed by Fourier analysis. Specific periodical patterns were found in various segments of the sequences. These characteristic periodicities vary within the same proteins as well as between spidroin 1 and spidroin 2 sequences. It is possible that alterations in periodicity help to recognize contact sites between the molecules. Spidroins of 2 type have similar sequence structure consisting of four parts with a particular periodical pattern. These parts are a constant C-terminal part, a long-periodical part, a short-periodical part, and a constant N-terminal part.

Amino Acid Sequence↗
PubMedDate not supplied

[An analysis of the secondary structure of spider spidroins I and II belonging to different species].

We have analyzed the secondary structure of spidroin proteins of I and II types, related to spiders of different species. We used standard methods of secondary structure prediction NNPREDICT and JPRED and also analyzed the occurrences of oligopeptides with a preferred secondary structure with the help of the OLIGON program. We have demonstrated that local segments of the polypeptide chain can adopt alpha- and beta-conformations as well as the left-handed helix of polyproline II type. Periodical patterns found in the amino acid distribution indicate that there is a possibility of development of a macroscopic order accompanied by local conformational transitions.

Amino Acid Sequence↗
PubMed2004

[Biological synthesis and purification of spider dragline silk protein polymers containing RGD three peptide].

Spider dragline silk is one of most perfect fibrous proteins in nature. As biomaterials, it has a wide application in tissue engineering due to its unique mechanical properties, good biocompatibility, slow degradation. In this paper, based on the highly repetitive sequence of spider dragline silk and with the introduced RGD peptide codons which involve cell adhesion, the DNA monomer sequence encoding RGD-spider dragline silk was synthesized, and then was used to construct the multimers by the strategy of "head to tail"; the multimers were ligated into prokaryotic expression vector pET-30a, and then the B121 (DE3) pLyS, were transformed the expression of recombinant protein was induced by the addition of IPTG. SDS-PAGE analysis shows that the molecular weight of products expressed here are 35KD and 60KD respectively in agreement with the desired. Western assay was used for determining the specification of products. Further, the purification process was groped for the producing of large quantity of synthetic proteins through high density fermentation.

Amino Acid Sequence↗
PubMed1992

Prelude activities in the synthesis of tissue-specific secretory protein products.

In studying the process of protein synthesis of a silk-producing organism we have found that several macromolecules must be synthesized in order for the process to occur. Through time course studies, we have found that small RNAs may play a paramount role in directing the finely orchestrated process. Alanine tRNA, U1 snRNA, and 5S RNA have been identified through Northern blotting as molecules timely and tissue-specific synthesized and upgraded as a prelude activity for the silk being produced.

Animals↗