Thermostable mannose-binding lectin from Dendrobium findleyanum with activities dependent on sulfhydryl content

A mannose-binding lectin was purified from Dendrobium (D.) findleyanum pseudobulb using mannan-agarose column chromatography. After heating in the presence of SDS with or without 2-mercaptoethanol on SDS-PAGE with a continuous gradient of 8%-20% acrylamide, the purified lectin showed only one protein band with a molecular mass of 14.5 kDa. Without heating, two bands were seen on the gel at the positions of 14.5 kDa and 53.7 kDa, but a higher amount of the 53.7 kDa protein was observed in the presence of 2-mercaptoethanol. Protein identification of both protein bands by liquid chromatography-tandem mass spectrometry showed three peptide fragments identical to parts of a lectin precursor from D. officinale; the lectin was named D. findleyanum agglutinin (DFA). Using various concentrations of native-PAGE and Ferguson plot, only one protein band revealed a molecular mass of 56.2 kDa, indicating four 14.5 kDa polypeptide subunits in the DFA. Isoelectric focusing revealed that the DFA had three conformational forms with an isoelectric point of 5.18, 4.87 and 4.72, whereas 2-mercaptoethanol-treated DFA showed only one band with an isoelectric point of 5.18. DFA exhibited specificity towards mannose using the solid-phase method. The binding activity, anti-fungal activity and hemagglutination activity of DFA were not affected by heat, but were increased by free sulfhydryl groups.     

Expression,purification, and characterization of recombinant Chinese shrimp crustin-like protein (CruFc) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A crustin-like protein (CruFc) from Fenneropenaeus chinensis was expressed in Pichia pastoris and then purified to electrophoretic homogeneity on a Sephacryl S-100 column with a band corresponding to the expected one (13 kDa) shown by 15% SDS-PAGE. Western blot indicated that the rCruFc specifically reacted with polyclonal rabbit anti-Fenneropenaeus chinensis CruFc. Production in a 5 l bioreactor gave 237 mg rCruFc/l. Antimicrobial assay revealed that 4 μM rCruFc inhibited growth of Staphylococcus aureus.     

Changes in nitrogen content and protein profiles following <Emphasis Type="Italic">in vitro</Emphasis> selection of NaCl resistant mung bean and tomato

Mung bean and tomato were in vitro selected on media containing 0, 25, 50, 100 and 150 mM NaCl. Two types of media (hormone supplemented media, CB and hormone free media, MS) were used for mung bean using cotyledon explants whereas two types of explants (cotyledons and shoot apices) were used for tomato on MS media. Total-N, protein content, nitrite reductase (NiR) activity and protein protein profiles were checked in selected plants and compared to original non selected ones. NaCl at low concentrations slightly increased total-N in shoots and roots of in vitro selected mung bean and tomato whereas higher concentrations induced significant reductions. Similar increases in protein content were detected at lower concentrations with no significant effects thereover. On the contrary, NaCl gradually inhibited NiR activity. Similar responses of total-N, protein and NiR activity, but with greater magnitudes, were detected in original plants. In addition, NaCl significantly reduced dry weights of shoots and roots of either in vitro selected or, in particular, original intact plants. Moreover, electrophoresis (SDS-PAGE) of protein from shoots of either in vitro selected or intact plants showed that NaCl induced new protein bands while some others were concomitantly disappeared. The induction of one or more of the 86.4, 79, 77.6, 77 and 71.5 kDa bands following in vitro selection and/or the disappearance of the 86 kDa band from intact plants seemed necessary for mung bean resistance. Also, the presence of 86.2 kDa band and/or the loss of the 85.8 and 57.5 kDa bands might be included in tomato resistance. Of these induced bands in mung bean selected on CB media, only two bands were detected in plants selected on MS media. In tomato, two bands lost following selection from cotyledons but only one band lost following selection from shoot apices. These changes in protein pattern therefore might serve as adaptive regulators for resistance to NaCl.     

Cloning,expression and immunogenicity of ferric enterobactin binding protein Fep B from <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7

The gene coding for ferric enterobactin binding protein from E. coli O157:H7 was amplifi ed. This gene was cloned and expressed as C-terminal His (6)-tagged protein. The SDS-PAGE analysis of the total protein revealed only two distinct bands, with molecular masses of 31kDa and 34kDa. The Ni-NTA chromatography purifi ed FepB and the osmotically shocked periplasmic fraction of IPTG induced cells showed only a single band of 31 kDa. Polyclonal mouse antibody was raised against the recombinant protein during 4 weeks after immunization. Western blot analysis of the recombinant FepB with mouse antiserum revealeda single band of 31 kDa. Identification and purification of FepB helped reveal its appropriate molecular mass. Polyclonal antibody raised against the recombinant protein reacted with bacterial FepB. The recombinant protein FepB could have a protective effect against E. coli O157:H7 and might be useful as an effective vaccine.     

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C, with a t ₅₀ of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase).     

A single replacement of histidine to arginine in EGFR-lytic hybrid peptide demonstrates the improved anticancer activity

Ribonucleotide reductase M1 (RRM1) is the regulatory subunit of the holoenzyme that catalyzes the conversion of ribonucleotides to 2′-deoxyribonucleotides. Its function is indispensible in cell proliferation and DNA repair. It also serves as a biomarker of therapeutic efficacy of the antimetabolite drug gemcitabine (2′,2′-difluoro-2′-deoxycytidine) in various malignancies. However, a mechanistic explanation remains to be determined. This study investigated how the alkylating agent N-ethylmaleimide (NEM) interacts with the inhibitory activity of gemcitabine on its target protein RRM1 in vivo. We found, when cells were treated with gemcitabine in the presence of NEM, a novel 110 kDa band, along with the 90 kDa native RRM1 band, appeared in immunoblots. This 110 kDa band was identified as RRM1 by mass spectrometry (LC–MS/MS) and represented a conformational change resulting from covalent labeling by gemcitabine. It is specific to gemcitabine/NEM, among 11 other chemotherapy drugs tested. It was also detectable in human tumor xenografts in mice treated with gemcitabine. Among mutations of seven residues essential for RRM1 function, C218A, C429A, and E431A abolished the conformational change, while N427A, C787A, and C790A diminished it. C444A was unique since it was able to alter the conformation even in absence of gemcitabine treatment. We conclude that the thiol alkylator NEM can stabilize the gemcitabine-induced conformational change of RRM1, and this stabilized RRM1 conformation has the potential to serve as a specific biomarker of gemcitabine’s therapeutic efficacy.     

Mannose-binding lectin from<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Rosc

A mannose-binding lectin was isolated from rhizomes of the medicinal plantCurcuma zedoaria. We used extraction with 20 mM phosphate buffer, ammonium sulfate precipitation, ion exchange chromatography on Q-Sepharose, gel filtration chromatography on Superdex 75, and reverse-phase HPLC. The purified lectin yielded a single band on SDS-PAGE that corresponded to a molecular mass of 13 kDa. This lectin exhibited hemagglutinating activity toward rabbit erythrocytes, which could be inhibited by mannose only. The lectin was digested with trypsin and its digests were analyzed using MALDI-TOF/TOF. Partial amino acid sequences were obtained from tandem mass spectra via automatedde novo sequencing, and were then identified by MS-BLAST homology searches to enable recognition of related proteins in other species. Inferred peptide sequences exhibited similarity to a mannose-binding lectin fromEpipactis helleborine, a member of the Orchidaceae.     

cDNA cloning and expression analysis of a mannose-binding lectin from <Emphasis Type="Italic">Pinellia pedatisecta</Emphasis>

Pinellia pedatisecta agglutinin (PPA) is a very basic protein that accumulates in the tuber of P. pedatisecta. PPA is a hetero-tetramer protein of 40 kDa, composed of two polypeptide chains A (about 12 kDa) and two polypeptides chains B (about 12 kDa). The full-length cDNA of PPA was cloned from P. pedatisecta using SMART RACE-PCR technology; it was 1146 bp and contained a 771 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acid residues with a 24 amino acid signal peptide. The PPA precursor contained 3 mannose-binding sites (QXDXNXVXY) and two conserved domains of 43% identity, PPA-DOM₁ (polypeptides A) and PPA-DOM₂ (polypeptides B). PPA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families such as Araceae, Alliaceae, Iridaceae, Liliaceae, Amaryllidaceae and Bromeliaceae. Southern blot analysis indicated that ppa belonged to a multi-copy gene family. Expression pattern analysis revealed that ppa expressed in most tested tissues, with high expression being found in spadix, spathe and tuber. Cloning of the ppa gene not only provides a basis for further investigation of its structure, expression and regulatory mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.     

AC29: the most abundant protein of Alexandrium catenella and its potential use in encystment/excystment studies

The objective of this research was to characterize specific protein(s) from Alexandrium catenella to evaluate its use as markers for specific physiological functions. To identify such protein(s) we concentrated our efforts on characterizing proteins with a high level of expression in vegetative cells of A. catenella. The electrophoretic analysis of a total protein cell extract showed the presence of a very abundant 29 kDa protein that we have named AC29. Analysis by 2D SDS-PAGE shows that the 29 kDa band contains one abundant protein (AC29) and various less abundant polypeptides, suggesting the presence of either different proteins with similar molecular weight or isoforms of AC29 protein. Ultracytolocalization using antibodies raised against gel purified AC29 indicates that this protein localizes within the chloroplast and that it is associated with thylakoid membranes, as well as with other membranes surrounding the chloroplast. Western blot analysis of cells grown under light starvation shows that the expression of the AC29 protein is down regulated. A similar analysis shows that this protein is not expressed in natural cysts or by isolated intracellular bacterium. The amino terminus of the AC29 protein that was recovered from 2D SDS-PAGE was sequenced. The sequence shows homology to the peridinin-chlorophyll a-protein from the marine organisms Alexandrium cohorticula, Amphidinium carterae and Symbiodinium. Based on these results, we suggest that the AC29 protein has the potential of being used as a marker for A. catenella encystment and excystment processes.     

Purification and characterization of levoglucosan kinase from <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis> YZ-215

A protein named as levoglucosan kinase (EC 2.7.-.-)was purified to homogeneity from a wild isolated strain of Lipomyces starkeyi YZ-215. The protein was purified approximately 30-fold by conventional ammonium sulphate fractionation followed by Resource Q chromatography and two steps of Superdex 200 chromatography, and its physical and kinetic properties were investigated. The purified enzyme showed a molecular weight of 48 kDa by SDS-PAGE and 47.7 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), respectively. The enzyme was stable at pH 7–10 and showed maximum activity at 30°C and pH 9.0. Kinetic constants (apparent K _m values) for levoglucosan and ATP were 68.6 ± 13.7 mM and 0.68 ± 0.06 mM, respectively. After in-gel digestion by trypsin, three peptides were sequenced and analyzed by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS). Data of the amino acid sequences indicated that this protein might be a novel kinase. The purification of levoglucosan kinase from L. starkeyi YZ-215 represented a fundamental step to provide insights into the efficient utilization of cellulosic pyrolysate by bioconversion.     

Purification and anti-fungal activity of chitinase against Pyricularia grisea in finger millet

Pseudomonas fluorescens isolate 1 (Pfl) protected finger millet plants treated with the ragi blast fungus, Pyricularia grisea, by upto 27% depending on the cultivar. Induction of pathogenesis-related proteins, viz., chitinase by Pfl isolate, was studied against Py. grisea. The activity of chitinase from plants treated with Pfl was significantly higher than the control plant after pathogen inoculation in all cultivars tested. Chitinase in the cultivars, with and without challenge by Py. grisea, revealed changes in the isoform pattern by western blot analysis. Chitinase was purified by affinity chromatography from the Pfl-treated leaves. It showed a single band at 57 kDa after SDS-PAGE. Western blot analysis using barley chitinase antiserum confirmed a 57 kDa chitinase. The chitinase had anti-fungal activity against Py. grisea in vitro. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of heat-shock response of the marine bacterium Vibrio harveyi

We have investigated heat-shock response in a marine bacterium Vibrio harveyi. We have found that 39 C was the highest tempature at which V. harveyi was able to grow steadily. A shift from 30° C to 39° C caused increased synthesis of at least 10 proteins, as judged by SDS-PAGE, with molecular masses of 90, 70, 58, 41, 31, 27, 22, 15, 14.5 and 14kDa. The 70, 58, 41 and 14.5 kDa proteins were immunologically homologous to DnaK, GroEL, DnaJ and GroES heat-shock proteins of Escherichia coli, respectively. V. harveyi GroES protein had a lower molecular mass (14.5 kDa) than E. coli GroES, migrating in SDS-PAGE as 15 kDa protein. We showed that a protein of ～43 kDa, immunologically reactive with antiserum against E. coli sigma 32 subunit (σ³²) of RNA polymerase, was induced by heat-shock and co-purified with V. harveyi RNA polymerase. These results suggest that the 43 kDa protein is a heat-shock sigma protein of V. harveyi. Preparation containing the V. harveyi sigma 32 homologue, supplemented with core RNA polymerase of E. coli, was able to transcribe heat-shock promoters of E. coli in vitro.     

Synthesis,Expression and Purification of a Type of Chlorotoxin-like Peptide from the Scorpion, <Emphasis Type="Italic">Buthus martensii</Emphasis> Karsch,and its Acute Toxicity Analysis

A gene, rBmK Cta, encoding a chlorotoxin-like peptide from the scorpion, Buthus martensii Karsch, was synthesized according to the sequence optimized for codon usage in Escherichia coli and was expressed in E.␣coli BL21 (DE3) using a pExSecI expression system in which the IgG-binding domain-ZZ of protein A is fused to the N-terminal of rBmK CTa. The fusion protein, ZZ-rBmK CTa, was expressed in soluble form (7.8 mg l⁻¹) and was purified to give a single band on SDS-PAGE. The domain-ZZ of fusion protein ZZ-rBmK CTa was removed by cleavage of an Asn–Gly peptide bond with hydroxylamine. The rBmK CTa was separated from the IgG-binding moiety by a second passage through the IgG affinity column. Western blot analysis demonstrated that this protein was rBmK CTa. Acute toxicity assay in mice demonstrated that the rBmK CTa had an LD₅₀ value of 4.3 mg kg⁻¹.     

High-level expression of a soluble snake venom enzyme,gloshedobin, in E. coli in the presence of metal ions

The mature gene of gloshedobin, a snake venom thrombin-like enzyme from the snake, Gloydius shedaoensis, was cloned and expressed in strain E. coli BL21(DE3). Having been induced by IPTG, the recombinant gloshedobin was in both soluble and insoluble forms. To avoid inclusion body formation, expression was optimized at 25 °C. Furthermore, a 50% increase in solubilization of the target protein was obtained by adding 0.1 mM Mg²⁺ to the medium. The purified recombinant gloshedobin gave a 44 kDa band on SDS-PAGE gel.     

Vitellin of the sweet potato whitefly,Bemisia tabaci: Biochemical characterization and titer changes in the adult

SDS-PAGE of the sweet potato whitefly (Bemisia tabaci) egg extract showed one major band (approximately 190 kDa) and two minor bands (approximately 75 kDa and 67 kDa). A distinct 190 kDa band was also present in male extract. On SDS gels the vitellin band of the greenhouse whitefly (Trialeurodes vaporarium) was larger, about 220 kDa. The native molecular mass of sweet potato whitefly vitellin was estimated to be 375 kDa using 4–20% native pore-limiting gel electrophoresis. Its isoelectric point was estimated to be 7.3 using isoelectric focusing. Two-dimensional gel electrophoresis and densitometry were used to estimate vitellin subunit composition; the data suggest that the sweet potato whitefly vitellin is likely to be a 380 kDa native molecule formed by two 190 kDa subunits. The two minor bands (75 kDa and 67 kDa) may be breakdown products of the native vitellin. This conclusion was supported by a Western blot of an SDS-PAGE gel of partially degraded female and egg extracts, which showed that polyclonal antiserum raised against the 190 kDa polypeptide recognized the 75 kDa and 67 kDa bands. Seven hybridoma cell lines secreting monoclonal antibodies against the 190 kDa band were screened, and one of them (S1A2G9H2) was mass produced. The antibody recognized the 190 kDa band in a Western blot. All the screened monoclonal antibodies were female and egg-specific by ELISA and/or Western blot, suggesting that the 190 kDa band in male extract was not a vitellin. A sensitive ELISA was established that could detect as little as 1/40 of an egg equivalent of vitellin using the monoclonal antibody from S1A2G9H2. Profiles of female sweet potato whitefly reproductive activities (egg laying, amount of vitellin in the female, and total vitellin produced by a female) within 2 days after eclosion were determined. Arch. Insect Biochem. Physiol. 34:223–237, 1997. © 1997 Wiley-Liss, Inc.     

A Rainbow Trout Lectin with Multimeric Structure

A novel lectin has been identified in rainbow trout serum and plasma. The lectin binds to Sepharose (an agarose polymer) in a calcium-dependent manner. Glucose, N-acetyl-glucosamine, mannose, N-acetyl-mannosamine, l-fucose, maltose and α-methyl-mannoside are good inhibitors of this binding, whereas glucosamine and d-fucose inhibits to a lesser degree and mannosamine and galactose do not inhibit the binding to Sepharose. When analysed by SDS-PAGE under non-reducing conditions, the lectin appears as a characteristic ladder of bands with approximately 16 kDa between consecutive bands. Upon reduction, the lectin appears as a 16-kDa band. On size-exclusion chromatography of trout serum and plasma, the protein emerges over a broad range corresponding to sizes from about 2000 kDa to less than 200 kDa. The NH₂-terminal sequence (AAENRNQXPPG) shows no significant homology with known proteins. Because of the characteristic appearance in non-reducing SDS-PAGE and the lectin activity, we propose to name the protein “ladderlectin.”     

Strawberry fruit protein with a novel indole-acyl modification

Achenes and receptacle tissue of Fragaria vesca, L. cultivar Yellow Wonder were shown to contain conjugated indole-3-acetic acid (IAA) that was not soluble in organic solvents and yielded IAA after strong alkaline hydrolysis, suggestive of IAA attached to plant proteins. This solvent insoluble conjugated IAA accounted for between 0.4 and 4 ng of IAA per gram fresh weight of tissue in both achenes and receptacles. To investigate this strawberry conjugate class further, a polyclonal antibody was produced to IAA–glycine attached to BSA that detected neutral indole acid esters, monocarboxylic-amino acid IAA conjugates and IAA proteins. Using immunoblotting, both achenes and receptacles of strawberry were shown to have primarily an immuno-detectable band at 76 kDa. Two-dimensional polyacrylamide gel electrophoresis yielded a wide band that was analyzed by LC–MS/MS analysis following in-gel trypsin digestion. Peptides derived from the immuno-detectable band were tentatively identified by peptide fragment analysis as being from either a chaperonin related to the hsp60 class of proteins or, alternatively, an ATP synthase. This is one of the first reports of an IAA modified protein in fruit tissue.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Characterization of an OmpA-like outer membrane protein of the acidophilic iron-oxidizing bacterium, <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

An OmpA family protein (FopA) previously reported as one of the major outer membrane proteins of an acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans was characterized with emphasis on the modification by heat and the interaction with peptidoglycan. A 30-kDa band corresponding to the FopA protein was detected in outer membrane proteins extracted at 75°C or heated to 100°C for 10 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the band was not detected in outer membrane proteins extracted at ≤40°C and without boiling prior to electrophoresis. By Western blot analysis using the polyclonal antibody against the recombinant FopA, FopA was detected as bands with apparent molecular masses of 30 and 90 kDa, suggesting that FopA existed as an oligomeric form in the outer membrane of A. ferrooxidans. Although the fopA gene with a sequence encoding the signal peptide was successfully expressed in the outer membrane of Escherichia coli, the recombinant FopA existed as a monomer in the outer membrane of E. coli. FopA was detected in peptidoglycan-associated proteins from A. ferrooxidans. The recombinant FopA also showed the peptidoglycan-binding activity.     

Cloning and characterization of the inulinase gene from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.     

Identification of the plasmid-encoded immunity protein for colicin El in the inner membrane of Escherichia coli

A set of plasmids containing portions of the Col El plasmid were transformed into recA⁻ cells. These cells, after UV irradiation, only incorporate labelled amino acids into plasmid-encoded proteins. UV-irradiated cells label a 14.5 kDa band if they are phenotypically immune to colicin E1, and do not contain this band if they are sensitive to colicin E1. We conclude that the 14.5 kDa protein is the colicin E1 immunity protein. When the inner and outer membranes of these cells are fractionated, the labelled band appears in the inner membrane. The immunity protein must be an intrinsic inner membrane protein, confirming the predictions made by hydrophobicity calculations from primary sequence data.MaxicellCol El plasmidImmunity proteinHydrophobicity calculation