Construct Synthetic Gene Encoding Artificial Spider Dragline Silk Protein and its Expression in Milk of Transgenic Mice

State Key Laboratory for Agrobiotechnology, China Agriculture University, Beijing, PR China.
Animal Biotechnology (Impact Factor: 0.76). 02/2007; 18(1):1-12. DOI: 10.1080/10495390601091024
Source: PubMed


Based on the known partial cDNA sequence of dragline silk protein an artificial gene monomer, a 360 bp sequence, was designed and polymerized to encode an analog of dragline silk protein. Six tandem copies of monomer were cloned into pBC1 vector and microinjected into the pronuclei of fertilized Kunming White eggs. Transgenic mice were screened by Polymerase Chain Reaction (PCR) and Southern blot which revealed that 10 mice (5 male, 5 female) among 58 mice were transgenic positive. Milk of five F0 mice and eight F1 mice was analyzed by Western blot, and two F0 mice and seven F1 mice expressed recombinant dragline silk protein. In transgenic mice milk a maximum of concentration of recombinant dragline silk protein was 11.7 mg/L by radioimmunoassay.

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    • "Consequently, scientists have turned to expressing spider silk proteins in transgenic systems, through recombinant DNA technologies. Spider silk proteins have been successfully expressed in different host systems, including Escherichia coli (Fahnestock & Irwin, 1997), yeast (Fahnestock & Bedzyk, 1997), transgenic animals such as goat, mouse (Xu et al., 2007; Lu et al., 2012), silkworms (Teule et al., 2012; Wen et al., 2010), and transgenic plants such as soybean, potato (Scheller et al., 2001) and tobacco (Scheller et al., 2001; Menassa et al., 2004). Unfortunately, these synthetic silk proteins have limited mechanical properties, compared to the pure natural silk. "
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    ABSTRACT: As an ancient arthropod with a history of 390 million years (Ma), spiders evolved numerous morphological forms resulted from adaptation to different environments. The venom and silk of spiders, which have promising commercial applications in agriculture, medicine and engineering fields, are of special interests to researchers. However, little is known about their genomic components, which hinders not only understanding spider biology but also utilizing their valuable genes. Here we report on deep sequenced and de novo assembled transcriptomes of three orb-web spider species, Gasteracantha arcuata, Nasoonaria sinensis and Gasteracantha hasselti which are distributed in tropical forests of south China. With Illumina paired-end RNA-seq technology, 54,871, 101,855 and 75,455 unigenes for the three spider species were obtained respectively, among which 9,300, 10,001 and 10,494 unique genes are annotated respectively. From these annotated unigenes, we comprehensively analyzed silk and toxin gene components and structures for the three spider species. Our study provides valuable transcriptome data for three spider species which previously lack any genetic/genomic data. The results have laid the first fundamental genomic basis for exploiting gene resources from these spiders. This article is protected by copyright. All rights reserved.
    Insect Science 10/2013; 21(6). DOI:10.1111/1744-7917.12068 · 2.14 Impact Factor
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    • "Recently, a native-sized dragline silk protein (284.9 kDa) was expressed in a modified E. coli strain, although the expression level was relatively low [5]. Several eukaryotic systems, including yeast, plants, and cultured insect or mammalian cells, can also be used to express recombinant spider silk proteins [6], [7], [8], [9]; however, protein expression in these systems has typically been limited by low yields and/or high cost. "
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    ABSTRACT: Spider silks are desirable biomaterials characterized by high tensile strength, elasticity, and biocompatibility. Spiders produce different types of silks for different uses, although dragline silks have been the predominant focus of previous studies. Spider wrapping silk, made of the aciniform protein (AcSp1), has high toughness because of its combination of high elasticity and tensile strength. AcSp1 in Argiope trifasciata contains a 200-aa sequence motif that is repeated at least 14 times. Here, we produced in E. coli recombinant proteins consisting of only one to four of the 200-aa AcSp1 repeats, designated W(1) to W(4). We observed that purified W(2), W(3) and W(4) proteins could be induced to form silk-like fibers by shear forces in a physiological buffer. The fibers formed by W(4) were ∼3.4 µm in diameter and up to 10 cm long. They showed an average tensile strength of 115 MPa, elasticity of 37%, and toughness of 34 J cm(-3). The smaller W(2) protein formed fewer fibers and required a higher protein concentration to form fibers, whereas the smallest W(1) protein did not form silk-like fibers, indicating that a minimum of two of the 200-aa repeats was required for fiber formation. Microscopic examinations revealed structural features indicating an assembly of the proteins into spherical structures, fibrils, and silk-like fibers. CD and Raman spectral analysis of protein secondary structures suggested a transition from predominantly α-helical in solution to increasingly β-sheet in fibers.
    PLoS ONE 11/2012; 7(11):e50227. DOI:10.1371/journal.pone.0050227 · 3.23 Impact Factor
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    • "These include another microbial system, Pichia (Fahnestock and Bedzyk 1997), mammalian cells (Lazaris et al 2002) and a plant system using transgenic tobacco (Menassa et al 2004). The alternative system that had previously attracted most interest is spider silk production in transgenic animals, including mouse (Xu et al 2007) and in dwarf Nigerian goats. "
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    ABSTRACT: New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation.
    Biomedical Materials 02/2012; 7(1):012002. DOI:10.1088/1748-6041/7/1/012002 · 3.70 Impact Factor
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