Cryoprotective effect of the serine-rich repetitive sequence in silk protein sericin.
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The silk proteins, fibroin and sericin, are produced in the silk gland of Bombyx mori, and hydrophilic sericin envelops fibroin with successive sticky layers in the formation of a cocoon. To study the biological functions of sericin, we focused on the serine-rich sericin peptide consisting of 38 amino acids, which is a highly conserved and internally repetitive sequence of a sericin protein. The corresponding gene was chemically synthesized, and the PCR-amplified gene was ligated to oligomerize sericin peptide and fused at the amino terminus to a His-tagged and proteolytic cleavage sequence in an inducible expression vector. When the dimers of sericin peptides were overexpressed in Escherichia coli, the transformants showed a prominent increase in cell viability after freezing in medium. Further, the purified dimeric sericin peptide from E. coli was found to be effective in protecting lactate dehydrogenase from denaturation caused by freeze-thaw. Both of these protective effects against freezing stress in cells and proteins were also observed with sericin hydrolysate. These results indicate that this unique sericin peptide, like sericin, has a high cryoprotective activity and will be valuable as a new biomaterial for industrial use. (+info)
Supplemental silk protein, sericin, suppresses colon tumorigenesis in 1,2-dimethylhydrazine-treated mice by reducing oxidative stress and cell proliferation.
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This study was done to discover the underlying mechanism of the inhibitory effect of sericin against colon tumorigenesis. Mice were fed a diet with 30 g/kg sericin for 115 d, and given a weekly injection of 1,2-dimethylhydrazine (10 mg/kg body weight) for the initial 10 wk. Dietary supplemental sericin caused a 62% reduction in the incidence of colonic adenoma (P<0.05), but did not affect the incidence of colonic adenocarcinoma. Sericin intake significantly reduced the number of colon adenomas. Consumption of sericin significantly reduced the BrdU labeling index of colonic proliferating cells and the expression of colonic c-myc and c-fos. The levels of colonic 8-hydroxydeoxyguanosine, 4-hydroxynonenal, and inducible nitric oxide synthase protein were significantly suppressed by sericin. The results suggest that dietary sericin suppresses the development of colon tumors by reducing oxidative stress, cell proliferation, and nitric oxide production. (+info)
Isolation of three main sericin components from the cocoon of the silkworm, Bombyx mori.
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To characterize the sericin components of the cocoon of silkworm Bombyx mori, fresh cocoon shells were dissolved in saturated aqueous lithium thiocyanate containing 2-mercaptoethanol, and fractionated by ethanol precipitation. Cocoon sericin was found to mainly consist of three polypeptides having molecular masses of the 400, 250, and 150 kDa estimated by SDS-PAGE, which corresponds to the sericin present in the middle, anterior, and posterior part of the middle silk gland. The amino acid compositions of the 400 and 150 kDa components were similar to each other, but that of the 250 kDa component was different. This suggests differences in the coding gene and properties of the 250 kDa sericin from the other two. (+info)
Cloning, expression, and assembly of sericin-like protein.
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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 beta-sheets during the dialysis. Congo red-stained recombinant sericin fibrils exhibited apple-green birefringence, indicating long-range order in the array of beta-sheets. Biosynthetic sericin has a high content of polar amino acids (e.g. > 40 mol % serine), leading to a beta-sheet conformation formed by hydrogen bonding via polar zipper interactions. Analysis of recombinant sericin sequence using Mandel-Gutfreund's (Mandel-Gutfreund, Y., and Gregoret, L. M. (2002) J. Mol. Biol. 323, 453-461) definition of polar and non-polar amino acids showed that the hydrophobicity pattern resembles the most frequent pattern of amyloidogenic proteins, polar amino acid aggregates (PPPPP). Many beta-proteins and peptides are designed to study amyloidogenesis using a polar/non-polar alternating pattern (PNPNPN). Sericin-like proteins or peptides provide an alternative model in terms of hydrophobicity pattern with which to explore questions related to beta-sheet formation and amyloidogenesis. The glue-like property of sericin is attributed to the hydrogen bonding between serine residues of sericin with serine residues in the fibroin structural components of silk fiber. (+info)
Characterization of sericin powder prepared from citric acid-degraded sericin polypeptides of the silkworm, Bombyx Mori.
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Acid-degraded sericin powder (AC-SP) was prepared from aqueous solution containing citric acid-degraded sericin polypeptides of Bombyx mori. The morphological and biochemical properties of AC-SP were compared with those of alkali-degraded sericin powder (AL-SP) and hot-water degraded sericin powder (HW-SP). Based on an SEM analysis, AC-SP showed a thin film structure of 10-100 microm with good dispersity while AL-SP and HW-SP had a much larger thin film structure (<500 microm). The extract of AC-SP showed stronger trypsin inhibitor activity due to cocoon shell trypsin inhibitor (CSTI-IV) than that of HW-SP. The extract of AL-SP showed no CSTI-IV activity. It was found that AC-SP was a trypsin inhibitor complex powder and that the release of CSTI-IV from AC-SP depended on pH and ion strength. Similar powder materials were obtained when such organic acids as tartaric acid and succinic acid were used. These results suggest that the acid-degraded sericin polypeptides work as a protein matrix to which CSTI-IV may bind ionically. (+info)
Sericin enhances attachment of cultured human skin fibroblasts.
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Human skin fibroblasts were cultured on sericin prepared from cocoon shells. The living cell number after 72 h was enhanced to 250% of the no-sericin control. The increase was due to the acceleration of the initial attachment of the cells. It was found that sericin M, the main component of about 400 kDa, and its serine-rich repetitive domain were the active principles. (+info)
Preparation of elastic silk sericin hydrogel.
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This paper reports a preparation method for silk sericin hydrogel using the Sericin-hope silkworm, whose cocoons consist almost exclusively of sericin. Sericin solution, prepared from Sericin-hope cocoons, contains intact sericin and forms elastic hydrogels with the addition of ethanol. The sericin hydrogel can be prepared without crosslinking by chemicals or irradiation and might be usable as a naturally occurring biomaterial. (+info)
Interfacial behavior of fatty-acylated sericin prepared by lipase-catalyzed solid-phase synthesis.
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Fatty-acylated sericin {1:0.7 molar ratio of sericin (Mr 18,700) to oleic acid} was prepared by lipase-catalyzed solid-phase synthesis in n-hexane containing oleic acid to endow sericin with interfacial properties. Acylation with oleic acid was confirmed by 1H-NMR. The fatty-acylated sericin exhibited superior emulsifying activity index and emulsion stability in the presence of 0-0.5 M NaCl, in a temperature range of 30-80 degrees C and pH range of 2-7, as compared with the control sericin. The fatty-acylated sericin (1:0.4 molar ratio) prepared by using low-molecular-weight sericin (Mr 5,000) also exhibited superior emulsifying properties. The affinity of the fatty-acylated sericin to a hydrophobic surface as evaluated by a biomolecular interaction analyzer was about twice as much as that of the control sericin. The fatty-acylated sericin showed retarded water vaporization, similar to the control sericin, indicating good retention of moistness, and was adsorbed four times as much to defatted wool with little desorption as compared with the control sericin. (+info)