Purification and characterization of bovine mannan-binding protein

Bovine mannan-binding protein (bMBP) was observed in serum byits Ca²⁺ -dependent binding to mannan and by an M_rof 28 kDaunder reducing conditions on sodium dodecyl sulphate—polyacrylamidegel electrophoresis (SDS—PAGE). The lectin was isolatedby precipitation with polyethyl-eneglycol (PEG), affinity chromatographyon mannan—Sepharose eluted with EDTA, and absorption onSepharose 4B rabbit anti-bovine Ig to remove anti-mannan antibodies.Fractions containing the lectin were reapplied to mannan—Sepharoseand eluted first with N-acetyl-D-glucosamine (GlcNAc) to removeconglutinin, and then with mannose to elute the 28 kDa lectin.Further purification was achieved by ion-exchange chromatographyon Mono-Q and by man-nose-gradient elution from a mannan-Sepharosecolumn. SDS—PAGE of the purified lectin showed three highmolecular weight bands under non-reducing conditions. The reducedprotein gave a single band of 28 kDa. On gel permeation chromatographyunder non-dissociating conditions, the protein emerged at avolume corresponding to M_r     

Comparative analysis of agglutinins from hemolymph and albumin gland ofHelix pomatia

Summary The hemolymph ofHelix pomatia contains a weak agglutinating activity. This lectin concentration was calculated to be about 1.8 g·ml^–1. Among the different red blood cells tested, pronase-treated sheep erythrocytes were found to be the most suitable indicator cells. Their agglutination could be inhibited by GalNAc and GlcNAc. The serum agglutinin was isolated by affinity chromatography using Sephadex G-200 as the matrix. It exhibited a single band in discontinuous PAGE. In the presence of SDS, subunits of 27000 daltons were obtained which, after addition of 2-mercaptoethanol, partly dissociated into 13000-dalton subunits. The biochemical properties observed were compared with those of the well-known blood group A-specific lectin from the albumin gland ofH. pomatia.Abbreviations GalNac N-acetyl-galactosamine - GlcNAc N-acetyl-glucosamine - SDS sodium dodecylsulfate     

Origin of a metal-binding protein in serum of Mytilus edulis

The most abundant humoral protein of the haemolymph of Mytilusedulis is known to bind a variety of heavy metals (Renwrantzet al., 1998 Comp. Biochem. Physiol. A, 121: 175–180).This serum protein band 1 (SPB1) was isolated from Mytilus serumby a two-step purification procedure. For the purified proteina molecular weight of 34 kDa was estimated under denaturingconditions and of 37 kDa in its reduced form. After blottingof Mytilus serum onto a nitrocellulose membrane, Cu-bindingby SPB1 was visualized. Furthermore, indications were obtainedof Ca-binding properties of SPB1 and of some bands postulatedto be SPB1-oligomers. Formation of dimers was confirmed by theirreaction with SPB1-specific antibodies. On immunoblots, anti-SPB1detected the metal-binding protein in postnuclear haemocyteextracts. After further fractionation of the cell homogenateby sucrose gradient centrifugation, the indicator antibodiesrecognized SPB1 in the cytosol, as a component of the plasmamembrane and in the fraction of granules which was assumed toinclude secretory vesicles. Investigation of the supernatantfrom a 24 h haemocyte culture indicated release of the metal-bindingprotein by blood-cells. Incubations of different haemocyte monolayerson slides with anti-SPB1 indicated expression of SPB1 in amountsof 55% to almost 100% of the cells, whereby the intensity ofantibody-staining varied between individual haemocytes. Intensitywas independent of cell type as density gradient separationof haemocytes into basophilic and eosinophilic granulocytesresulted in similar staining patterns in both groups. Thus,all granulocytes seemed to be able to produce SPB1 with a varyingdegree of activity indicating a hitherto unknown regulationsystem. (Received 4 June 2007; accepted 6 September 2007)     

Purification and characterization of a mucin-binding mycelial lectin from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> with potent mitogenic activity

Mucin-specific lectin from mycelium of Aspergillus nidulans was purified using anion exchange and gel filtration chromatographic techniques with an overall recovery of 32% and 21.97-fold purification. The purified lectin migrated as a single band in SDS–PAGE with an apparent molecular mass of 34 kDa. Carbohydrate analysis revealed that it is a glycoprotein with total sugar content of 2.54%. Optimal agglutination was observed when serially diluted lectin was incubated with human type O erythrocyte suspension at pH 7.0–8.0 and temperature 20–30°C. Lectin was found to be completely stable within pH 5.0–8.0 and temperature at or below 40°C. Demetallization by extensive dialysis against EDTA did not alter its haemagglutination activity. Lectin activity was reduced to half after 24 h incubation with urea and thiourea, with no such effect of guanidine HCl. The lectin showed potent mitogenic response towards mouse splenocytes, attaining a maximum at 200 μg/ml as compared to untreated control cells. Mitogenic lectins are invaluable tools to assess the functioning of immune cells. None of the microfungal lectin has yet been investigated for mitogenic activity. This is the first report on mitogenic activity of lectin from Aspergillus sp.     

Conversion and degradation of shellfish wastes by <Emphasis Type="Italic">Serratia</Emphasis> sp. TKU016 fermentation for the production of enzymes and bioactive materials

A chitosanase and a protease were purified from the culture supernatant of Serratia sp. TKU016 with shrimp shell as the sole carbon/nitrogen source. The molecular masses of the chitosanase and protease determined by SDS–PAGE were approximately 65 and 53 kDa, respectively. The chitosanase was inhibited completely by Mn²⁺, but the protease was enhanced by all of tested divalent metals. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitosanase and protease were (pH 7, 50°C, pH 6–7, <50°C) and (pH 8–10, 40°C, pH 5–10, <50°C), respectively. SDS (2 mM) had stimulatory effect on TKU016 protease activity. The result demonstrates that TKU016 protease is SDS-resistant protease and probably has a rigid structure. Besides, TKU016 culture supernatant (2% SPP) incubated for 2 days has the highest antioxidant activity, the DPPH scavenging ability was about 76%. With this method, we have shown that shrimp shell wastes can be utilized and it’s effective in the production of enzymes, antioxidants, peptide and reducing sugar, facilitating its potential use in biological applications and functional foods.     

Purification of Lectin from Micropropagated Roots Derived from Aseptic Seedling of <Emphasis Type="Italic">Canavalia ensiformis</Emphasis> L.

A well-organized protocol has been developed for high frequency root germination from the seed of Canavalia ensiformis on Murashige and Skoog (MS) medium. Surprisingly, the seeds that were grown on the MS medium having no growth hormone showed the best response. Roots of 30 days old aseptic seedling were homogenized and a lectin from them was purified on Sephadex G-50 affinity column. The finding that final product is a pure lectin was confirmed by specific hemagglutinating property. The final root lectin yield was 0.6% and eluted as a single peak. Root lectin specific activity was 50 times more than the seed lectin. Sugar specificity activity by hemagglutination-inhibition assay indicated that lectin belongs to glucose/mannose-specific group. Interestingly, the lectin was found to be 25 kDa, similar to molecular mass of Concanavalin A purified from seed of C. ensiformis, as revealed by SDS–PAGE. Thus, Concanavalin A from either source can be used for development of transgenic crops that are capable of expressing lectin gene and hence can efficiently perform biological nitrogen fixation by giving rise to nodules in their root. The advantage of this method is that purification of Concanavalin A in tissue culture conditions is easier, handy and is less time consuming.     

Heterogeneity and subunit composition of the haemoglobins of five tilapiine species (Teleostei, Cichlidae) of the genera Oreochromis and Sarotherodon

Haemoglobins of five tilapiine species of the genera Oreochromis and Sarotherodon were investigated. By gel filtration chromatography a molecular weight of 67–69 kDa was determined for the tetrameric molecules which remained stable between pH 5.0 and pH 9.1. When subjected to sodium dodecyl sulphate-Urea-polyacrylamide gel electrophoresis (PAGE), haemoglobins of all species each were split into monomers of three different molecular weights ranging between 16.3 kDA and 17.6 kDa. Subsequently, isoelectric focusing separated haemolysates into about 23 differently charged tetrameric haemoglobins that were arranged in species-specific patterns. This diversity was shown to result from the occurrence of different types of globin chains. By acidic urea PAGE a total of seven major α-globins and five major β-globins were detected and species-characteristic chain variants were identified. To determine the globin chain composition of particular haemoglobin tetramers, 26 bands were isolated by isoelectric focusing and analysed by acidic urea PAGE. Tetramers consisted of doublets of identical α- and identical β-chains (α₂β₂, symmetric tetramers), or combinations of three (α₂ββ*; αα*β₂) or four (αα*ββ*) distinct chains (asymmetric tetramers). Finally, globin chains of Oreochromis niloticus were subjected to partial N-terminal amino acid sequencing. Differences in the composition of the three major β-chains could be shown, whereas the α-chains were N-terminally blocked. Accepted: 12 September 1997     

Erratum

Free oligosaccharide regulation during mammalian protein N-glycosylation doi:10.1093/glycob/cwn003 Glycobiology vol. 18 no. 3 pp. 210–224, 2008 Figure 3 and its     

Phytase produced on citric byproducts: purification and characterization

Citric pulp is an agro-industrial residue from the citrus processing industry with low inorganic phosphorus content applied in animal feed. A new bioprocess was developed to produce and purify a new phytase generated on citric pulp fermentation by Aspergillus niger FS3. The phytase was purified by cationic-exchange, anionic-exchange chromatography and chromatofocusing steps. From SDS–PAGE analysis, the molecular weight of the purified phytase was calculated to be 108 kDa. The phytase had an optimum pH of 5.0–5.5 and an optimum temperature of 60°C. The phytase displayed high affinity for phytate, and the K _m was 0.52 mM. The purified phytase was sufficiently able to withstand pelleting temperatures, retaining sufficiently high phytate-degrading activity.     

<Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: occurrence in the air and clinical significance

Trichoderma harzianum, a filamentous fungus, is being widely used as a potential biopesticide. The potential of this fungus in causing skin sensitization, however, was poorly investigated as yet. The objective of this study was to monitor the occurrence of T. harzianum in the air and to explore its skin sensitizing potential. Seasonal periodicity of T. harzianum was studied for the years 2002–2004 by an Andersen air sampler. The skin sensitizing potential of T. harzianum extract was studied in 389 patients with suspected respiratory allergy by skin prick test (SPT) and specific IgE level was determined by ELISA. SDS–PAGE and immunoblotting were also performed. T. harzianum colony count varied from 3.69 to 134.88 CFU m⁻³ with the peak achieved in February. Relative humidity was found to be a significant (P < 0.05) factor predicting the occurrence of T. harzianum in the air. Positive skin reaction (wheal diameter ≥ 3 mm) was observed in 105 patients (26.99%). T. harzianum crude extract was resolved in 18 protein bands (12–72 kDa) on SDS–PAGE (12% gel) including two IgE-binding protein bands (21 and 32 kDa). T. harzianum can be considered an important inhalant allergen.     

Cloning and expression of a highly active recombinant alkaline phosphatase from psychrotrophic Cobetia marina

Alkaline phosphatase catalyzes the hydrolysis of phosphomonoesters and is widely used in molecular biology techniques and clinical diagnostics. We expressed a recombinant alkaline phosphatase of the marine bacterium, Cobetia marina, in Escherichia coli BL21 (DE3). The recombinant protein was purified with a specific activity of 12,700 U/mg protein, which is the highest activity reported of any bacterial alkaline phosphatase studied to date. The molecular mass of the recombinant protein was 55–60 kDa, as determined by SDS–PAGE, and was observed to be a dimer by gel filtration analysis. The enzyme was optimally active at 45°C and the recombinant alkaline phosphatase efficiently hydrolyzed a phosphoric acid ester in luminescent and fluorescent substrates. Therefore, this enzyme can be considered to be extremely useful as a label conjugated to an antibody.     

3D-Garden: a system for modelling protein-protein complexes based on conformational refinement of ensembles generated with the marching cubes algorithm

Motivation: Reliable structural modelling of protein–proteincomplexes has widespread application, from drug design to advancingour knowledge of protein interactions and function. This workaddresses three important issues in protein–protein docking:implementing backbone flexibility, incorporating prior indicationsfrom experiment and bioinformatics, and providing public accessvia a server. 3D-Garden (Global And Restrained Docking ExplorationNexus), our benchmarked and server-ready flexible docking system,allows sophisticated programming of surface patches by the uservia a facet representation of the interactors’ molecularsurfaces (generated with the marching cubes algorithm). Flexibilityis implemented as a weighted exhaustive conformer search foreach clashing pair of molecular branches in a set of 5000 modelsfiltered from around 340 000 initially. Results: In a non-global assessment, carried out strictly accordingto the protocols for number of models considered and model qualityof the Critical Assessment of Protein Interactions (CAPRI) experiment,over the widely-used Benchmark 2.0 of 84 complexes, 3D-Gardenidentifies a set of ten models containing an acceptable or bettermodel in 29/45 test cases, including one with large conformationalchange. In 19/45 cases an acceptable or better model is rankedfirst or second out of 340 000 candidates. Availability: http://www.sbg.bio.ic.ac.uk/3dgarden (server) Contact: v.lesk{at}ic.ac.uk Supplementary information: Supplementary data are availableat Bioinformatics online. Associate Editor: Burkhard Rost     

Evidence for a lactose-mediated association between two nuclear carbohydrate-binding proteins

Nuclear proteins were extracted in 2 M NaCl from membrane-depletednuclei isolated from HL60 cells. Extracted proteins were submittedto affinity chromatography columns containing immobilized glucose,galactose or lactose. The polypeptides present in the differenteluted fractions were resolved by SDS—PAGE and were eithersilver stained or analysed by immunoblotting with monoclonalor polyclonal antibodies, respectively, raised against the glucose-bindingprotein CBP67 and the galactose-binding proteins CBP35 and L14.The results presented here show that HL60 cell nuclei containCBP35 and a glucose-binding lectin of 70 kDa (CBP70). Thesedata account for the previously reported binding of neoglyco-proteinscontaining glucosyl and galactosyl residues to HL60 cell nuclei.Furthermore, the present study provides the new informationthat CBP35 can associate with CBP70 by interactions dependenton the binding of CBP35 to lactose, and the results of someaffinity chromatography experiments strongly suggest that CBP35and CBP70 associate by protein—protein interactions. Thepotential function of this lactose-mediated interaction is discussedwith respect to data recently reported by others showing thatCBP35 is involved in in vitro mRNA splicing and that lactoseinhibits the processing of the pre-RNA substrate. HL60 lectins nucleus protein—protein interactions     

O-Linked fucose in glycoproteins from Chinese hamster ovary cells

We report our discovery that many glycoproteins synthesizedby Chinese hamster ovary (CHO) cells contain fucose in O-glycosidiclinkage to polypeptide. To enrich for the possible presenceof O-linked fucose, we studied the lectin-resistant mutant ofCHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferaseI and are therefore unable to synthesize complex-type N-linkedoligosaccharides. Lec1 cells were metabolically radiolabelledwith [6-³H]fucose and total glycoproteins were isolated. Glycopeptideswere prepared by proteolysis and fractionated by chromatographyon a column of concanavalin A (Con A)— Sepharose. Thesets of fractionated glycopeptides were treated with mild base/borohydrideto effect the ß-elimination reaction and release potentialO-linked fucosyl residues. The ß-elimination produced[³H]fucitol quantitatively from [³H]fucose-labelled glycopeptidesnot bound by Con A-Sepharose, whereas none was generated bytreatment of glycopeptides bound by the lectin. The total [³H]fucose-labelledglycoproteins from Lec1 cells were separated by SDS—PAGEand detected by fluorography. Treatment of selected bands ofdetectable glycoproteins with mild base/borohydride quantitativelygenerated [³H]fucitol. Pretreatment of the glycoproteins withN-glycanase prior to the SDS—PAGE method of analysis causedan enrichment in the percentage of radioactivity recovered as[³H]fucitol. Trypsin treatment of [³H]fucose-labelled intactCHO cells released glycopeptides that contained O-linked fucose,indicating that it is present in surface glycoproteins. Thesefindings demonstrate that many glycoproteins from CHO cellscontain O-linked fucosyl residues and raise new questions aboutits biosynthesis and possible function. fucose glycoproteins monosaccharide O-linked     

Purification and characterization of dye degrading of veratryl alcohol oxidase from Pseudomonas aeruginosa strain BCH

In the present study we have purified the intracellular veratryl alcohol oxidase (VAO) enzyme from Pseudomonas aeruginosa strain BCH to evaluate its dye decolorizing potential. The enzyme was purified by ion exchange chromatography using DEAE cellulose followed by gel filtration chromatography using Biogel P-100. The molecular weight of the purified enzyme was estimated by polyacrylamide gel electrophoresis (PAGE) analysis. The VAO was purified up to 12 and 16.3-fold by ion exchange and gel filtration chromatography respectively. VAO was estimated to be about 85 kDa by SDS–PAGE. The optimum pH and temperature for purified VAO was 3 and 55°C respectively. The purified enzyme exerted its optimal activity with veratryl alcohol and also oxidized various other substrates, whereas diminished activity was noted in case of tryptophan and xylidine. The metal ions Mn⁺⁺ and Hg⁺⁺ were found to suppress the oxidase activity. The purified enzyme decolorized different dyes with variable decolorization rates and efficiencies. Decolorization mechanism of Remazol Black by purified enzyme was studies in detail using various analytical techniques like HPLC, GC–MS and FTIR. This study is useful for understanding the precise role of Pseudomonas aeruginosa strain BCH in the decolorization of textile dyes containing industrial wastewater.     

Purification and properties of aminoaldehyde dehydrogenase from <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5′-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS–PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a pI value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that Avena AMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K _m values for APAL, ABAL, and GBAL were 1.5×10^–6, 2.2×10^–6, and 1.3×10^–5 M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH. Electronic Publication     

Codon optimization of the calf prochymosin gene and its expression in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Chymosin as an important industrial enzyme widely used in cheese manufacture. The yeast Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, low yields (80 U/ml in shake flask cultures) were obtained when the K. lactis strain GG799 was used to express chymosin. We hypothesized that the codon-usage bias of the host may have resulted in inefficient translation and chymosin production. To improve expression efficiency of recombinant calf chymosin in K. lactis strain GG799, we designed and synthesized a DNA sequence encoding calf prochymosin using optimized codons, while keeping the G + C content relatively low. We altered 333 nucleotides to optimize codons encoding 315 amino acids. In shaking flask culture, chymosin activity was 575 U/ml in the strain expressing the optimized gene, a sevenfold higher expression level compared with the non-optimized control. SDS–PAGE analysis revealed that the purified recombinant calf chymosin had a molecular mass of 35.6 kDa, the same as the molecular weight of native calf chymosin. Alpha-casein, beta-casein, and kappa-casein were incubated with the recombinant calf chymosin from K. lactis strain GG799 or chymosin from calf stomach and the breakdown products were analyzed by SDS–PAGE. Both the recombinant calf chymosin and the native calf chymosin specifically hydrolyzed kappa-casein. Our results show that codon optimization of the calf chymosin gene improves expression in K. lactis strain GG799. Genetic manipulation to optimize codon usage has important applications for industrial chymosin production.     

Expression of a staphylokinase,a thrombolytic agent in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A gene encoding staphylokinase from Staphylococcus aureus was cloned into the plant transformation binary vector pCAMBIA 1304. The transgene was introduced into the genome of A. thaliana via in planta Agrobacterium tumefaciens–mediated genetic transformation. The presence of the staphylokinase gene was confirmed by PCR in 60% of the investigated plants. The presence of the fusion protein (119 kDa) was confirmed by SDS–PAGE and Western blot analysis in protein extracts from putative transgenics. Furthermore, the amidolytic assay confirmed the activity of SAK in protein extracts in 23 out of 45 transgenic lines of A. thaliana plants.     

The Bark Lectin of Robinia pseudoacacia: Purification and Partial Characterization

A lectin was purified from the bark of Robinia pseudoacaciaby sequential ion-exchange chromatography on DEAE-Sepharoseand CM-Toyopearl. The purified lectin was estimated to havea molecular weight of 106 kDa and to be a homotetramer of subunitswith a molecular weight of 29 kDa. Antibodies raised againstthe bark lectin cross-reacted with a 29-kDa polypeptide duringWestern blot analysis, showing that the antibodies are specificfor the bark lectin. The antibodies against the lectin fromRobinia bark cross-reacted with polypeptides in extracts ofthe seeds and bark of Sophora japonica, indicating that thelectin from Robinia bark is immunologically related to the lectinsof Sophora. However, the antibodies did not cross-react withproteins from Robinia seeds and leaves. The first twenty aminoacid residues from the N-terminus of the lectin from Robiniabark were determined and compared with those of the Sophoralectins. (Received July 13, 1991; Accepted December 12, 1991)