A novel method for dissolution and stabilization of non-mulberry silk gland protein fibroin using anionic surfactant sodium dodecyl sulfate

The importance of silk protein has increased because of its potential use as a natural biopolymer for tissue engineering and biomedical applications. In this report we show a novel and ecofriendly method for dissolution of gland silk protein fibroin. Non-mulberry silk fibroin from mature fifth instar larvae of Antheraea mylitta was found to be optimally soluble in 1% (w/v) anionic surfactant sodium dodecyl sulfate (SDS). Regenerated fibroin showed distinct bands of approximately 395 and 197 kDa on electrophoresis in non-reducing and reducing conditions, respectively. Enhanced fibroin dissolution via internalization of hydrophobic amino groups inside a hydrophilic amino acid core in the form of micelles was observed. Prolonged storage stability without gelation of SDS-extracted fibroin was seen. Atomic force microscopy showed micellar aggregation with mean micellar aggregation size of 8 nm. Circular dichroism spectroscopy revealed predominantly helical conformation due to surfactant addition with internal protein conformational changes as revealed by fluorescence spectroscopic studies.     

Affinity purification and partial characterization of IgM-like immunoglobulins of African catfish, Clarias gariepinus (Burchell, 1822)

IgM like macroglobulin from bovine serum albumin (BSA)-immunized African catfish C. gariepinus was purified by affinity chromatography and partially characterized. The molecular weight of this macroglobulin was 840 kDa, as estimated by gel filtration chromatography. Purified macroglobulin was analyzed using SDS-PAGE under reducing and non-reducing conditions. The molecular weight (MW) of heavy and light chain was 74.8 kDa and 27.2 kDa respectively, in presence of a reducing agent. In non-reducing SDS-PAGE, a single high MW band was observed representing tetrameric form.     

Isolation, purification and characterization of silk protein sericin from cocoon peduncles of tropical tasar silkworm, Antheraea mylitta

A high molecular weight water-soluble glue protein, sericin was identified in the cocoon peduncle (a strong thread connecting the cocoons to the branches of the tree with a ring) of the tropical tasar silkworm, Antheraea mylitta. The sericin was isolated by 8 M urea containing 1% sodium dodecyl sulfate and β-mercaptoethenol (2%) or by 1% sodium chloride. The protein was purified by gel filtration chromatography. In SDS-PAGE, a single band of approximately 200 kDa was detected both in non-reducing and reducing conditions. Amino acid analysis showed that the protein is enriched in glycine and serine. There is a slight difference observed in amino acid composition between the sericin from cocoon peduncle and cocoon of A. mylitta. Secondary structure estimation by circular dichroism spectrometry showed 36.7% β-sheets, 52.7% random coils, 10.6% turns and no helices.     

Purification and properties of cysteine proteinase inhibitors from rabbit skeletal muscle

Two cysteine proteinase inhibitors, CPI-L and CPI-H, were purified from rabbit skeletal muscle by means of successive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. The molecular mass of CPI-L was 13 kDa on gel permeation chromatography and SDS-PAGE under reducing conditions and was 15 kDa on SDS-PAGE under non-reducing conditions. The molecular mass of CPI-H was 23 kDa on gel permeation chromatography and it was converted to 13 kDa by SH-reducing agent. Although CPI-H showed single protein band with 13 kDa on SDS-PAGE under reducing conditions, it showed four protein bands with 21, 20, 15 and 13 kDa on SDS-PAGE under non-reducing conditions. Therefore, CPI-H was suggested to have a complicated subunit structure for which S-S bonds and some non-covalent bonds would be responsible. CPI-L and CPI-H were stable in the range of pH 3.0-9.5 and up to 80 degrees C. CPI-L and CPI-H were suggested to inhibit cathepsins B, H and L by a non-competitive mechanism. The inhibition constants (Ki) of CPI-L and CPI-H showed that both CPIs have much higher affinity against cathepsins H and L than against cathepsin B.     

Isolation and Biochemical Characterization of a Galactoside Binding Lectin from <Emphasis Type="Italic">Bauhinia variegata</Emphasis> Candida (BvcL) Seeds

A new lectin (BvcL) from seeds of a primitive Brazilian Caesalpinoideae, the Bauhinia variegata candida was purified and biochemical characterized. BvcL was isolated by gel filtration chromatography on Sephadex G75 and affinity chromatography on immobilized d-lactose column. SDS-PAGE showed that BvcL under non-reducing condition presents two bands of 68 and 32 kDa and a single band of 32 kDa in reducing condition. However, only one band was seen in native PAGE. The hemagglutination activity of BvcL was not specific for any human blood group trypsin-treated erythrocytes. Carbohydrate inhibition analysis indicated that BvcL is inhibited by lactose, galactose, galactosamine and other galactoside derivates. Amino acid analysis revealed a large content of Ser, Gly, Thr, Asp and Glu and low concentrations of Met, Cys and His. Intrinsic fluorescence of BvcL was not significantly affected by sugar binding galactose; and aromatic-region CD is unusually high for plant lectins. The N-terminal amino acid sequence of 17 residues showed 90% sequential homology to galactose-specific legume lectins of the subfamily Caesalpinoideae.     

A novel mitogenic and antiproliferative lectin from a wild cobra lily, Arisaema flavum

A novel lectin having specificity towards a complex glycoprotein asialofetuin was purified from tubers of Arisaema flavum (Schott.) by affinity chromatography on asialofetuin-linked amino-activated silica beads. A. flavum gave a single peak on HPLC size exclusion and a single band on non-denatured PAGE at pH 4.5. The molecular mass of the lectin, as determined by gel filtration chromatography, was 56 kDa. In SDS-PAGE, pH 8.3, the lectin migrated as a single band of 13.5 kDa, under reducing and non-reducing conditions, indicating the homotetrameric nature. A. flavum lectin (AFL) readily agglutinated rabbit, rat, sheep, goat, and guinea pig erythrocytes but not human ABO blood group erythrocytes even after neuraminidase treatment. This lectin is stable up to 55 degrees C and does not require metal ions for its hemagglutination activity. AFL was completely devoid of sulphur containing amino acids and was rich in aspartic acid and glycine. In Oucterlony's double immunodiffusion, the antisera raised against A. flavum lectin showed distinct lines of identity with those of other araceous lectins. AFL showed potent mitogenic activity towards BALB/c splenocytes and human lymphocytes in comparison to Con A, a well-known plant mitogen. AFL also showed significant in vitro antiproliferative activity towards J774 and P388D1 murine cancer cell lines.     

Renaturation, purification, and characterization of human truncated macrophage colony-stimulating factor expressed in Escherichia coli

A human truncated macrophage colony-stimulating factor (M-CSF) encoding the amino acid residues from 3 to 153 of the native M-CSF was expressed by using a two-cistron expression system in Escherichia coli. The truncated M-CSF found in inclusion bodies was renatured and had CSF activity. Purification, which included a QAE-ZeTa preparative cartridge concentration step followed sequentially by HPLC on TSK-gel Phenyl-5PW and TSK-gel DEAE-5PW columns, gave an overall yield of 63.8%. The purified truncated M-CSF had a specific activity of 4 x 10(7) units/mg of protein. Peptide mapping of a lysylendopeptidase digest by reversed-phase HPLC confirmed the amino acid sequence predicted from the cDNA sequence. SDS-PAGE of the purified truncated M-CSF gave a single band at 17 kDa under reducing conditions and at 32 kDa under non-reducing conditions. Activated Thiol-Sepharose 6B column chromatography and other experiments failed to detect any free cysteine residue in spite of the existence of 7 cysteine residues in the truncated M-CSF subunit. These results indicate that it is a dimeric structure linked by one or more intermolecular disulfide bonds.     

Vitellogenin-derived yolk proteins in a hybrid sturgeon, bester (Huso huso x Acipencer ruthenus): identification, characterization and course of proteolysis during embryogenesis

Vitellogenin (Vg) and its corresponding yolk protein (YP) products, YP1, YP2 and YP3, were isolated from serum of estrogen-treated hybrid sturgeon (bester; Huso huso X Acipencer ruthenus) and eggs from untreated fish, respectively. Vitellogenin had an apparent molecular mass of 580 kDa and appeared as two major bands corresponding to 180 kDa and 120 kDa after SDS-PAGE. Apparent molecular weights of YP1, YP2 and YP3 were 370 kDa, 88 kDa and 19 kDa, respectively. After SDS-PAGE, YP1 appeared as a main band of 110 kDa, while YP2 was resolved as a single band of 94 kDa and 29 kDa band under non-reducing and reducing conditions, respectively. Yolk protein 3 appeared as a diffuse band corresponding to 16 kDa and two faint bands below 14.4 kDa after SDS-PAGE. However, the 16 kDa band alone was observed after dephosphorylation with alkaline phosphatase. The course of cleavage of yolk proteins in bester embryos and alevins was observed by SDS-PAGE and Western blotting from fertilization onward. After hatching, the main 110 kDa band of YP1 was degraded into smaller peptides during development, while YP2 hardly showed any such structural changes. The amino acid compositions of purified yolk proteins indicated that YP1, YP2 and YP3 were bester lipovitellin, beta-component, and phosvitin, respectively.     

Neutral proteinase inhibitors in PMN leukocytes. II. Isolation and characterization of a neutral proteinase inhibitor from porcine PMN leukocytes

An inhibitor of neutral proteinases was purified from porcine PMN leukocytes by gel filtration on Sephadex G-75 superfine and ion-exchange chromatography on Mono S. Thus an inhibitor preparation with a specific inhibitory activity against chymotrypsin of 10 IU/mg was obtained. In dodecyl sulfate gel electrophoresis a single protein band with an apparent molecular mass of 40 kDa was found under reducing conditions. Under non-reducing conditions the inhibitor forms higher molecular mass aggregates. On isoelectric focusing several protein bands with isoelectric points between pH 7.0 and 7.5 could be separated. The amino-acid composition of the inhibitory protein was determined. The inhibition mechanism was studied and association rate constants (kon) were measured and calculated for the reaction with chymotrypsin as well as leukocyte and pancreatic elastase. In Western blot analysis and in enzyme immunoassay studies crossreactivity between antibodies directed against porcine leukocyte neutral proteinase inhibitor and the corresponding inhibitor of bovine PMN leukocytes could be demonstrated.     

A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.     

Isolation and characterization of a lectin from Grifola frondosa fruiting bodies

An N-acetylgalactosamine-specific lectin (GFL) was isolated from Grifola frondosa fruiting bodies by affinity chromatographies on acid-treated Sepharose CL-4B and then GalNAc-Toyopearl. The isolated lectin agglutinated all types of erythrocytes equally. Molecular masses estimated by gel filtration under various buffers and matrices varied from 30 to 52 kDa. On the other hand, SDS-PAGE in the presence or absence of 2-mercaptoethanol showed three major bands of 33, 66 and 100 kDa and a faint band of 65 kDa. This lectin exhibited GalNAc-specificity. The protein was a glycoprotein containing 3.3% total sugar, and the amino acid analysis revealed a high content of acidic and hydroxy amino acids and a low content of methionine and histidine. GFL was cytotoxic against HeLa cells. The toxicity did not appear after preincubating the lectin with the haptenic sugar N-acetylgalactosamine.     

β-Galactosidase from Ginkgo biloba seeds active against β-galactose-containing N-glycans: purification and characterization

In this study, we purified an acidic β-galactosidase to homogeneity from Ginkgo biloba seeds (β-Gal’ase Gb-1) with approximately 270-fold purification. A molecular mass of the purified β-Gal’ase Gb-1 was estimated about 35 kDa by gel filtration and 32 kDa by SDS-PAGE under non-reducing condition, respectively. On the other hand, β-Gal’ase Gb-1 produced a single band with a molecular mass of 16 kDa by SDS-PAGE under reducing condition. The N-terminal amino acid sequences of 32 kDa and 16 kDa molecules were the same and identified as H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-, suggesting that β-Gal’ase Gb-1 may function as a homodimeric structure in vivo. When complex-type N-glycans containing β-galactosyl residues were used as substrates, β-Gal’ase Gb-1 showed substantial activity for β1-4 galactosyl residue and modest activity for β1-3 galactosyl residue with an optimum pH near 5.0. Based on these results, the involvement of β-Gal’ase Gb-1 in the degradation of plant complex-type N-glycans is discussed.     

Purification, N-terminal amino acid sequence, and some properties of Cu, Zn-superoxide dismutase from Japanese flounder (Paralichthys olivaceus) hepato-pancreas

Cu, Zn-superoxide dismutase (SOD) has been purified to homogeneity from Japanese flounder Paralichthys olivaceus hepato-pancreas. The purification of the enzyme was carried out by an ethanol/chloroform treatment and acetone precipitation, and then followed by column chromatographies on Q-Sepharose, S-Sepharose and Ultrogel AcA 54. On SDS-PAGE, the purified enzyme gave a single protein band with molecular mass of 17.8 kDa under reducing conditions, and showed approximately equal proportions of 17.8 and 36 kDa molecular mass under non-reducing conditions. Three bands were obtained when the purified enzyme was subjected to native-PAGE, both on protein and activity staining, but the electrophoretic mobility of the purified enzyme differed from that of bovine erythrocyte Cu, Zn-SOD. Isoelectric point values of 5.9, 6.0 and 6.2, respectively, were obtained for the three components. The N-terminal amino acid sequence of the purified enzyme was determined for 25 amino acid residues, and the sequence was compared with other Cu, Zn-SODs. The N-terminal alanine residue was unacetylated, as in the case of swordfish SOD. Above 60°C, the thermostability of the enzyme was much lower than that of bovine Cu, Zn-SOD.     

Amino acid sequence and characterization of C-type lectin purified from the snake venom of Crotalus ruber

Galactoside-binding lectin was purified from the snake venom of Crotalus ruber by affinity chromatography on a lactose-agarose column, and the complete amino acid sequence was determined. The C. ruber venom lectin (CRL) showed a single band of 28 kDa by SDS-polyacrylamide electrophoresis under non-reducing conditions, but it showed a single band of 15 kDa under reducing conditions, indicating that CRL is a disulfide-linked homodimer of 15 kDa subunit. CRL specifically recognized beta-galactosides such as thiodigalactoside followed by N-acetylgalactosamine when examined with their inhibitory effects on CRL-induced hemagglutination. A CRL subunit was composed of 135 residues containing nine Cys residues and showed a high similarity to other C-type galactoside-binding lectins from snake venoms. C. atrox lectin (CAL) showed almost the same sequence except for eight amino acid residues. Neither CRL nor CAL induced platelet aggregation by itself or inhibited platelet aggregation mediated by von Willebrand factor or fibrinogen with agonists. CRL showed a similar oligomeric form and the sugar specificity as CAL, but it showed different divalent cation sensitivity such as Mn(2+) and Ni(2+). Homology modeling suggested that the amino acid substitution found in CRL does not affect sugar recognition of the lectin but might alter the conformation and influence the sugar binding pocket induced by the metal-ion binding.     

Purification and biochemical characterization of a 70 kDa sericin from tropical tasar silkworm, Antheraea mylitta

Sericin isolated from the cocoon of the tropical tasar silkmoth Antheraea mylitta showed three major bands, with the lowest 70 kDa. This band was purified by anion exchange chromatography. Immunoblotting with concanavalin-A suggests a glycoprotein and CD analysis of secondary structure includes beta-sheet. Amino acid analysis shows that the protein is enriched in glycine and serine while the mole percentages of these two amino acids are different from sericin of mulberry silkworm. An anti A. mylitta sericin antibody was able to cross-react with sericin from A. assamensis but not the sericin of Bombyx mori and Philosamia ricini. Immunoblot analysis with proteins isolated from middle silk gland of A. mylitta at different developmental stages of larva showed that the 70 kDa sericin is developmentally regulated. These data extend the range of biochemical features found in this unusual family of proteins and may help in developing an improved understanding of their role in forming environmentally stable fibroin fiber-sericin composite structures (cocoons).     

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from the edible mushroom Schizophyllum commune

An N-acetyl-D-galactosamine (GalNAc)-specific lectin was purified from the edible mushroom, Schizophyllum commune, using affinity chromatography on a porcine stomach mucin (PSM)-Sepharose 4B column. Under reducing and non-reducing conditions, SDS-polyacrylamide gel electrophoresis gave a major band of 31.5 kDa. The Schizophyllum commune lectin (SCL) showed high affinity toward rat erythrocytes and the sugar inhibition assay exhibited its sugar specificity highly toward lactose and N-acetyl-D-galactosamine. It was stable at 55 degrees C for 30 min and at pH 3-10 for 18-h test. The lectin was shown to be a glycoprotein with cytotoxic activity against human epidermoid carcinoma cells. The N-terminus of SCL was blocked but amino acid sequences of internal tryptic peptides showed moderately sequence similarities with some other fungal and plant lectins. Crystals of SCL were obtained by the sitting drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant, and gave an X-ray diffraction pattern to approximately 3.8 angstroms resolution.     

Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb （Amaryllidaceae）

A novel mannose-bindlng aggiutinln was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% （NH4）2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel flitration on Sephscryl S-100. The purified Z. candida agglutlnln （ZCA） migrated as a single band of 12 kDa on sodium dodecyi suifate-poiyecryiamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the iectln, as datermlned by gel filtration chromatography, was 48 kDa. The results Indicated that ZCA was composed of four Identical subunlts of 12 kDa each （homotetramerlc nature）. The ZCA agglutlhated rabbit erythrocytes, Escherichla coil and Saccharomyces cerevislae ceils at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bloassays Indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was 〉 90% at 0.26%. A degenerate primer was designed In accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3＇- and 5＇-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA Includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptlde, a mature protein, and a C-terminal cleavage amino acids sequence. Molecular modeling of ZCA Indicated that Its three-dimensional atructure strongly resembies that of the snowdrop aggiutinin. Blocks＇ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes （QDNY）.     

Isolation of an N-acetyl-D-glucosamine specific lectin from the rhizomes of Arundo donax with antiproliferative activity

A lectin with antiproliferative activity towards human cancer cell lines and mitogenic towards human peripheral blood mononuclear cells was purified from the rhizomes of Arundo donax (Linn.) by affinity chromatography on N-acetyl-d-glucosamine linked to epoxy-activated sepharose-6B. The pure preparation apparently yielded a single band of approximately 15 kDa on SDS-PAGE, pH 8.3, under both reducing and non-reducing conditions. The molecular mass of native lectin was 32 kDa as determined by gel filtration chromatography. This showed the lectin to be a dimer, with subunits not held together by disulphide linkages. The A. donax lectin (ADL) agglutinated rabbit erythrocytes and the agglutination was inhibited by N-acetyl-d-glucosamine and its di- and trimer. The lectin was thermostable upto 55 degrees C and showed optimum activity in the range of pH 7.0-9.0 and comprised of 2.1% carbohydrate content.     

Isolation and characterization of a lectin from Grifola frondosa fruiting bodies

An N-acetylgalactosamine-specific lectin (GFL) was isolated from Grifola frondosa fruiting bodies by affinity chromatographies on acid-treated Sepharose CL-4B and then GalNAc-Toyopearl. The isolated lectin agglutinated all types of erythrocytes equally. Molecular masses estimated by gel filtration under various buffers and matrices varied from 30 to 52 kDa. On the other hand, SDS-PAGE in the presence or absence of 2-mercaptoethanol showed three major bands of 33, 66 and 100 kDa and a faint band of 65 kDa. This lectin exhibited GalNAc-specificity. The protein was a glycoprotein containing 3.3% total sugar, and the amino acid analysis revealed a high content of acidic and hydroxy amino acids and a low content of methionine and histidine. GFL was cytotoxic against HeLa cells. The toxicity did not appear after preincubating the lectin with the haptenic sugar N-acetylgalactosamine.     

Purification and characterization of an invertase produced by Aspergillus ochraceus TS.

Purification and characterization of an extracellular invertase produced by Aspergillus ochraceus TS are reported. The enzyme was purified (42-fold) from culture filtrate by salt precipitation, ion-exchange and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single band of molecular mass 66 kDa. The molecular mass of the native enzyme was found to be 130 kDa by gel filtration. The purity of the protein was also checked against its antiserum raised in rabbits by two-dimensional immunodiffusion in agarose gel and Western blot that showed a single band. It is a glycoprotein with mannose as its carbohydrate residue. The enzyme showed high affinity for sucrose with a Km of 3.5 mM. The amino acid analysis revealed a high proportion of acidic residues but it had a low content of cysteine, histidine and arginine comparable to other fungal invertases.