Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk

Fukushima, Yasumasa

doi:10.1002/(sici)1097-0282(19980405)45:4<269::aid-bip1>3.0.co;2-j

Cited by 63 publications

(42 citation statements)

References 25 publications

(1 reference statement)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The oligonucleotides can be ligated to constitute silk genes expressible in bacteria ( Figure 2 and see below). [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] Another approach is based on expressing authentic spider cDNA in optimized hosts other than bacteria that can be produced at large scale. Furthermore, several eukaryotic hosts are able to ''decode'' the codon usage of spider genes, are not size-limited in gene expression, and have a low frequency of gene recombination.…”

Section: Why Is Spider Silk a Prime Target For Biomaterials Researchers?supporting

confidence: 78%

Biotechnological Production of Spider‐Silk Proteins Enables New Applications

Vendrely

Scheibel

2007

Macromolecular Bioscience

218

212

View full text Add to dashboard Cite

The outstanding mechanical properties of spider silks have motivated many researchers to establish biotechnological production techniques which are necessary to provide sufficient amounts of silk proteins for industrial applications. Based on recent developments in genetic engineering, two strategies for the recombinant production of spider-silk proteins have been established which are discussed in detail. Further, protein-design strategies are described, enabling the combination of silk properties with additional biological, chemical, or technical features. We highlight the potential of engineered and recombinantly-produced spider-silk proteins to provide the basis for a new generation of biomaterials.

show abstract

Section: Why Is Spider Silk a Prime Target For Biomaterials Researchers?supporting

confidence: 78%

Biotechnological Production of Spider‐Silk Proteins Enables New Applications

Vendrely

Scheibel

2007

Macromolecular Bioscience

218

212

View full text Add to dashboard Cite

show abstract

“…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).…”

mentioning

confidence: 99%

Cloning, Expression, and Assembly of Sericin-like Protein

Huang

Valluzzi

Bini

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

“…The cyanogen bromide cleavage of the hybrid proteins was performed according to the procedure described by Fukushima et al 24 Approximately 40 mg of purified hybrid His 6 -SLP protein was dissolved in 10 mL of 99% formic acid and diluted to 70% formic acid with distilled water. The mixture by added 100 mg of CNBr crystals was stirred at room temperature for 24 h and transferred into a dialysis membrane with a molecular weight cutoff of 3500.…”

Section: Cleavage Of Hybrid Proteins With Cyanogen Bromidementioning

confidence: 99%

“…Optimal codons for each amino acid can be selected using codon usage in E. coli. 24 Since the polypeptide expression level and the stability of the DNA itself are influenced by the base composition and sequence of the DNA, the codons were selected as many as A-T contents as possible. The DNA multiers were inserted into Nhe I and Spe I digested pUC118-linker, flanked by an ATG codon encoding methionine at each site in order to obtain the periodic portion of SLP by cleavage of the expressed hybrid proteins with cyanogens bromide (CNBr).…”

Section: Construction Of Monomeric and Multimeric Slp Dnasmentioning

confidence: 99%

Design, Expression, and Structural Characterization of Hybrid Proteins of Samia cynthia ricini and Bombyx mori Silk Fibroins

Asakura

Kato

Yao

et al. 2002

Polym J

View full text Add to dashboard Cite

ABSTRACT:Genetically engineering strategies have been applied to obtain the designed proteins [GGAGSGYGG-GYGHGYGSDGG(GAGAGS) 3 ] n of varying chain lengths (n = 2, 4 or 6); SLP2, SLP4, and SLP6, respectively. The amino acid sequence of the monomeric unit represents the incorporation of the consensus repeat sequences of the glycinerich region of Samia cynthia ricini followed by the repetitive crystalline region of Bombyx mori silk fibroins. The oligonucleotides were designed to encode the desired monomeric unit of silk-like hybrid proteins, which were then polymerised and expressed in Escherichi coli system. The monomer, SLP1, was synthesized chemically using solid phase method. The recombinant proteins were identified by N-terminal amino-acid sequencing and Western blotting. The structural characterizations of SLP1 and SLP6 samples were performed using 13 C CP/MAS NMR in the solid state. The structure of SLP1 in the solid state was distorted β-turn by judging from the chemical shift and line shape of the C β carbon of Ala residue, and the structural transition from distorted β-turn to β-sheet occurred partially in SLP6. The solution structure was studied in hexafluoro-2-propanol (HFIP) using CD method. Although both samples, SLP1 and SLP6, showed indication of the ordered structures, marked change in the band intensities was observed between two CD spectra. Especially, the CD pattern of SLP6 is in agreement with the pattern with right-handed 3 10 -helical conformation. KEY WORDS Silk-like Hybrid Proteins / Genetically Engineered Proteins / 13 C Cross Polarization Magic Angle Spinning NMR / Circular Dichroism / B.ṁori and S. c. ricini Silk Fibroins / Natural silks have become the subject of extensive study as model systems for high-performance biopolymers and polymer composites in part because of their impressive mechanical properties of the fibers. 1 In addition to high quality textiles, silks have been recognized to have not only excellent enzyme immobilization properties but also suggested to find potential applications in design and development of various biotechnological and biomedical devices. [2][3][4][5][6][7][8][9][10] The unique amino acid composition and the primary structure of the silk fibroin appear to be important 9 despite the fact that there are wide variations in the primary and secondary structures among different silks. For example, a well-known fibrous protein from the domesticated silkworm, B. mori, exhibits the amino acid composition (in mol%) as 42.9% Gly, 30.0% Ala, 12.2% Ser, 4.8% Tyr, and 2.5% valine. 9 On the other hand, the amino acid composition of the silk fibroin from a wild silkworm, S. c. ricini, is quite different. While the sum of Gly and Ala residues (82%) is similar to B. mori (73%) silk, the relative proportions of these residues are reversed (48.4% Ala and 33.2% Gly) in S. c. ricini. 11,12 An analysis of the amino acid sequence of B. mori silk fibroin reported by Zhou et al.,13 revealed the presence of unusual highly repetitive GAGAGS sequence motifs, comprising about 5...

show abstract

Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk

Cited by 63 publications

References 25 publications

Biotechnological Production of Spider‐Silk Proteins Enables New Applications

Biotechnological Production of Spider‐Silk Proteins Enables New Applications

Cloning, Expression, and Assembly of Sericin-like Protein

Design, Expression, and Structural Characterization of Hybrid Proteins of Samia cynthia ricini and Bombyx mori Silk Fibroins

Contact Info

Product

Resources

About