Construction, Cloning, and Expression of Synthetic Genes Encoding Spider Dragline Silk

Prince, John T.; McGrath, Kevin P.; DiGirolamo, Carla M.; Kaplan, David L.

doi:10.1021/bi00034a022

Cited by 270 publications

(225 citation statements)

References 38 publications

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“…Because the His-tag is designed at the C-terminal of the protein, no truncated target protein will be detected. The same issue has been observed previously in expression of spider silk genes (17,22). Overall yield of purified SRCn was 15-36 mg/l depending on the molecular weight of SRCn.…”

Section: Construction Of Recombinant Expression Plasmid Pet21a(ϩ)-srcn-supporting

confidence: 78%

“…Proteins derived from genetic engineering are characterized by control over composition, sequence, molecular weight, and stereochemical purity. Motifs of silkworm and spider silk proteins have been studied in a similar manner to understand structures in different environments, and spider silk proteins have been modified for the purpose of controlled assembly (17)(18)(19)(20)(21)(22)(23)(24).…”

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confidence: 99%

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Cloning, Expression, and Assembly of Sericin-like Protein

Huang

Valluzzi

Bini

et al. 2003

Journal of Biological Chemistry

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Recombinant sericin proteins of different molecular masses (17.4, 31.9, and 46.5 kDa), based on the 38-amino acid repetitive motif of native sericin, were cloned, expressed, and purified. The recombinant sericin self-assembled during dialysis (starting concentration of 2.5 mg/ml) forming twisted fibers. Circular dichroism and Fourier transform infrared spectroscopy studies demonstrated protein conformational transitions occurred from random coil to ␤-sheets during the dialysis. Congo red-stained recombinant sericin fibrils exhibited applegreen birefringence, indicating long-range order in the array of ␤-sheets. Biosynthetic sericin has a high content of polar amino acids (e.g. Silk of Bombyx mori is composed of two kinds of proteins: the fibroin which is synthesized in the posterior silk gland and the sericins produced by the middle silk gland. The sericins bind two fibroin fibers together in the anterior region of the gland as the fibers are spun to form the cocoon. Sericins represent a family of proteins whose molecular mass ranges from 20 to 310 kDa (2, 3) and are characterized by an unusually high serine content, 38.1 mol % (4). Sericins are soluble in hot water, which makes it possible to degum the silk fibroin threads.Two genes encode sericins, Ser1 and Ser2 (5-8). The different molecular weight sericins are the products of different splicing events at the transcript level. Some conformation studies with sericins from the silk gland of B. mori or regenerated sericin from B. mori cocoons suggested random coil with some ␤ structure (9, 10). However, the samples used in these studies were mixtures of the various native sericin proteins, therefore the structure of the individual proteins is unknown and the contribution of specific sequences toward secondary structure is not confirmed. Sericin model peptides such as poly(L-serine) (11-16), poly(O-benzyl-L-serine) (12), and poly(O-acetyl-L-serine) (13,14) were synthesized for structural feature characterization. The ability of poly(L-serine) to fold into a ␤ conformation depended on molecular weight. Low molecular weight poly(L-serine) (molecular weight 650) was soluble and adopted a random coil conformation in aqueous solution, whereas high molecular weight poly(L-serine) (molecular weight 105,000) formed ␤-sheets and was insoluble in water (11). However, homopolypeptides like poly(L-serine) do not match native sericin in terms of amino acid composition, sequence, and molecular weight. Therefore, to better understand sericin structure, interactions between sericin and fibroin, and the biological relevance of sericin in fiber structure, high molecular weight pure sericin-like proteins are required.Sericin 1 encoded by Ser 1 consists of 70 repeats of the 38-amino acid motif (5): SSTGSSSNTDSNSNSVGSSTS-GGSSTYGYSSNSRDGSV. The molecular mass of sericin 1 proteins is 76 -284 kDa. The 38-amino acid repeat motif exhibits a high content of hydrophilic amino acids: 44.7% serine, 10.5% threonine, and 7.9% tyrosine, very close to the average amino acid composition of serici...

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Section: Construction Of Recombinant Expression Plasmid Pet21a(ϩ)-srcn-supporting

confidence: 78%

mentioning

confidence: 99%

Cloning, Expression, and Assembly of Sericin-like Protein

Huang

Valluzzi

Bini

et al. 2003

Journal of Biological Chemistry

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“…2) by using cloning strategies previously described (38)(39)(40). The amino acid sequences of the two spider dragline silk fusion proteins with silicification-inducing domains, with (type 1) and without (type 2) a CRGD cell-binding motif, are shown in Fig.…”

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confidence: 99%

Novel nanocomposites from spider silk–silica fusion (chimeric) proteins

Foo

Patwardhan

Belton

et al. 2006

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

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Silica skeletal architectures in diatoms are characterized by remarkable morphological and nanostructural details. Silk proteins from spiders and silkworms form strong and intricate self-assembling fibrous biomaterials in nature. We combined the features of silk with biosilica through the design, synthesis, and characterization of a novel family of chimeric proteins for subsequent use in model materials forming reactions. The domains from the major ampullate spidroin 1 (MaSp1) protein of Nephila clavipes spider dragline silk provide control over structural and morphological details because it can be self-assembled through diverse processing methods including film casting and fiber electrospinning. Biosilica nanostructures in diatoms are formed in aqueous ambient conditions at neutral pH and low temperatures. The R5 peptide derived from the silaffin protein of Cylindrotheca fusiformis induces and regulates silica precipitation in the chimeric protein designs under similar ambient conditions. Whereas mineralization reactions performed in the presence of R5 peptide alone form silica particles with a size distribution of 0.5-10 m in diameter, reactions performed in the presence of the new fusion proteins generate nanocomposite materials containing silica particles with a narrower size distribution of 0.5-2 m in diameter. Furthermore, we demonstrate that composite morphology and structure could be regulated by controlling processing conditions to produce films and fibers. These results suggest that the chimeric protein provides new options for processing and control over silica particle sizes, important benefits for biomedical and specialty materials, particularly in light of the all aqueous processing and the nanocomposite features of these new materials.biomaterials ͉ nanostructures ͉ silaffin ͉ biomineralization ͉ ceramics C omplex mineralized composite systems in nature provide rich ground for insight into mechanisms of biomineralization and novel materials designs (1-4). Some of the more common sources of inspiration include seashells, insect exoskeletons, extracellular matrices involved in bone and other hard tissues, and biosilica skeletons. The formation of natural inorganic-organic composites is a multistep process, including the assembly of the extracellular matrix, the selective transportation of inorganic ions to discrete organized compartments with subsequent mineral nucleation, and growth delineated by preorganized cellular compartments. Silica skeletons found in nature are based on nanoscale composites wherein the organic components, usually proteins, are functional parts of the skeletal structures while also serving as silica-forming components (5, 6). As a result, materials' toughness is improved, strength is retained, and fine morphological control is achieved, all hallmark attributes of biological composites.Silica is widespread in biological systems and serves different functions, including support and protection in single-celled organisms, such as diatoms through to higher plants and animals (7,8...

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“…The recombinant silk protein showed a band corresponding to a molecular weight of B27 kDa (Figures 3a and b), which was roughly in accordance with the theoretical molecular weights of 22 kDa, considering that silk-based polymers generally do not run true to size on SDS-PAGE gels because of the hydrophobic nature of the protein. 25 On the basis of the results shown in Figures 3a and b, an incubation time of 3 or 4 h was considered optimal to express the silk protein. Therefore, an incubation of 4 h after the addition of IPTG was used for the expression of silk protein and PHB synthases throughout these experiments.…”

Section: Resultsmentioning

confidence: 99%

“…[17][18][19][20][21][22][23][24] Cloning and expression of native and synthetic silks have been achieved in various host systems using synthetic oligonucleotide versions of consensus repeats or variants of these repeats garnered from sequence data from native genes. [25][26][27][28] In this study, dual biosynthesis of both PHB and silk protein was designed and performed by double transformation into Escherichia coli to add biological functions to PHB-based biomaterials (Figure 1). This dual biosynthesis enabled a single-step preparation of PHB-based materials that exhibited several desirable biological functions.…”

Section: Introductionmentioning

confidence: 99%

Dual biosyntheses of poly[(R)-3-hydroxybutyric acid] and silk protein for the fabrication of biofunctional bioplastic

Numata

Doi

2011

Polym J

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This is the first report on dual synthesis of poly[(R)-3-hydroxybutyric acid] (PHB) and protein polymer by recombinant microorganisms for use in the fabrication of a biofunctional PHB-based material hybridized with functional proteins. PHB and recombinant silk protein, a model protein developed in this study, were synthesized by a recombinant Escherichia coli system, and then films of PHB hybridized with the silk protein (PHB/Silk) were prepared. The presence of the silk protein in the amorphous phase of the PHB/Silk film was demonstrated based on wide-angle X-ray diffraction and differential scanning calorimetry measurements, which also revealed the enhanced tensile strength and elongation at break of the PHB/Silk film. The cell adhesion of human mesenchymal stem cells (hMSCs) for the PHB/Silk film increased because of the addition of silk molecules to the film. This might have been because the silk molecules existed in the amorphous phase at the surface of the PHB/Silk films, and were exposed to hMSCs. The overall results illustrate the potential of this dual synthesis system as a versatile and useful platform method for preparation of biofunctional PHB-based materials, especially for biomedical applications.

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Construction, Cloning, and Expression of Synthetic Genes Encoding Spider Dragline Silk

Cited by 270 publications

References 38 publications

Cloning, Expression, and Assembly of Sericin-like Protein

Cloning, Expression, and Assembly of Sericin-like Protein

Novel nanocomposites from spider silk–silica fusion (chimeric) proteins

Dual biosyntheses of poly[(R)-3-hydroxybutyric acid] and silk protein for the fabrication of biofunctional bioplastic

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