Isolation of laminin by affinity chromatography on immobilized Griffonia simplicifolia I lectin (original) (raw)
International Journal of Pharmacy and Pharmaceutical Sciences, 2016
Objective: To isolate, identify and quantify lectin protein contents in Chamerion angustifolium L. dried raw material species namely; leaves in the flowering stage, buds, and flowers and the study of its activity. Methods: Lectin activity has been determined using the biological method called ratuserytroagglutination. This method is based on the formation of aggregates of lectins and rat erythrocytes using the floor amount of lectins that agglutinate erythrocytes as a unit of measurement. Additionally, the protein contents of the extracts have been determined using the Bradford assay method. Results: lectins activity from Chamerion angustifolium L. in leaves in the flowering stage, buds, and flowers were 2.72±0.06, 0.24±0.008, and 0.56±0.014 units/mg protein, respectively. The greatest lectins activity was in the leaves in the flowering stage followed by flowers and then in buds. Protein contents in leaves in the flowering stage, buds, and flowers were 4.71±0.03, 6.77±0.02, and 5.76±0.14 mg/ml, respectively. Conclusion: All proteins obtained from the Chamerion angustifolium L. plant raw material were shown to possess rat erythrocytes agglutinating activity. The crude extract of leaves in the flowering stage exhibited the strongest hemagglutinating activity of about 2.72 units/mg proteins, whereas the buds showed the lowest activity of about 0.24 units/mg proteins. It should be highlighted herein, although many plant lectins mimic the behavior of plant storage proteins, these lectins should not be classified as storage proteins.
.Isolation Purificationand Characterizationofa Lectinfrom Ocimumbasilicum Seeds OBS
Lectins are defined as the carbohydrate-binding proteins of non-immune origin that can interact and precipitates glycoconjugates from their solutions. Due to these features, many applications have been discovered for these proteins. The present investigation has been devoted to optimising a protocol for the extraction, purification and characterization of a lectin from the seeds of the medicinal plant Ocimum basilicum (OBSL). Different extraction procedures were employed, including varying buffers with variable pHs. Precipitation of the seeds' crude protein extract with salting-out using solid ammonium sulphate (AmS) at 40, 60 and 80% concentrations resulted in the segregation of the lectins into at least three isoforms. The lectin was purified to apparent electrophoretic homogeneity by fetuin-agarose affinity resin. The lectin was a trimer composed of three subunits of different molecular weights. Scanning the three AmS precipitated protein fractions at the UV ranges, indicates that the protein is rich in tryptophan rather than tyrosine. The lectin haemagglutination pattern studies showed that OBSL had a better affinity for the B+ human blood group, sheep and rabbit erythrocytes as compared to other human and animal blood types. The lectin was not inhibited by any simple sugar indicating its classification as a lectin with complex sugar specificity. OBSL was stable within the pH range from 2.5 to 10.5 showing two pH activity optima. It was optimally active from 40 to 50 °C. This study represents the first-ever report on this plant lectin and might stimulate further studies to extensively characterize this protein.
Purification of a lectin from Amaranthus leucocarpus by affinity chromatography
Phytochemistry, 1988
A lectin of M, ca 45,000 per subunit from Amaranthus leucocarpus seeds, has been isolated and purified by affinity chromatography using a stroma column. It is a glycoprotein (10% w/w carbohydrate) containing six N-acetyl-D-glucosamines, four D-galactoses, one D-glucose and traces of xylose residues for each three D-mannose residues per molecule. Its amino acid composition reveals a predominance of acidic residues (aspartic and glutamic) and of glycine and alanine. In addition, the lectin contains an unusual amount of essential aminoacids such as methionine, tryptophan and lysine. Electrophoretically and chromatographically homogenous, it focuses as a multiple-band protein in the pH range of 4.8-5.2. It agglutinates the different human blood groups of the ABO system equally well, albeit being inhibited by N-acetyl-D-galactosamine in a specific fashion. In contrast to A. caudatus hemagglutinin A. leucocarpus lectin is inhibited by serum glycoproteins such as fetuin, it is mitogenic and is not toxic.
Protein Expression and Purification, 2003
Hen eggs represent an easily available and inexpensive source of glycoproteins expressing a variety of sugars. Egg glycoproteins might therefore be exploited to purify by affinity chromatography carbohydrate-binding proteins (lectins) with different specificities. A method to generate an affinity matrix from hen eggs is described. The matrix was assayed for its ability to purify in a single step biologically active phytohemagglutinin, wheat germ agglutinin, lentil lectin, and peanut agglutinin. Milligrams of purified lectins per gram of matrix was obtained, with the only exception of peanut agglutinin that was not efficiently retained into the affinity column. Hen egg chromatography is a relatively simple, fast, and reproducible method to purify high amount of plant lectins.
Purification and characterization of three galactose specific lectins from Mulberry seeds (Morussp.)
European Journal of Biochemistry, 2001
Three lectins were extracted and purified from mulberry seeds by gel filtration of 100% ammonium sulfate saturated crude protein extract followed by ion-exchange chromatography on DEAE and CM-cellulose. The lectins were found to be homogeneous as judged by polyacrylamide disc gel electrophoresis. The molecular masses of the lectins as determined by gel filtration were 175 000 for MSL-1, 120 000 for MSL-2 and 89 500 for MSL-3. MSL-1 is dimer in nature, with the two monomers held together by disulfide bond(s), while MSL-2 and MSL-3 contain four nonidentical subunits that are held together by nonionic hydrophobic interactions. The lectins agglutinated rat red blood cells and this agglutination was inhibited specifically by galactose, methyl-aD -galactopyranoside, methyl-b-D-galactopyranoside, lactose and raffinose. The lectins MSL-1, MSL-2 and MSL-3 contained 5.7, 5.4 and 4.5% neutral sugars, respectively, and the sugar composition of the lectins was glucose and mannose for MSL-1 and galactose for both MSL-2 and MSL-3. The lectins exhibited strong cytotoxic effect in brine shrimp lethality bioassay.
Isolation and Purification of Lectin from Soybean (Glycine Max) Mahabeej Js-335
Journal of Pharmacognosy and Phytochemistry, 2016
Plant lectins have a wide spectrum of biological significance. In this project work the isolation and purification of the lectins (SBL) from the Soybean (Glycine max) Mahabeej JS-335 have been reported. The lactose binding lectins were purified from the Soybean seeds by performing affinity chromatography using a lactamil sephadex 100G. Affinity products were dialysed against PBS and SDS-PAGE was done to identify the fractions ‘molecular weight. This work explored different techniques of protein (lectin) purification and describing their characteristics.
The Plant Journal, 2003
A novel lectin has been isolated and cloned from leaves of Glechoma hederacea (ground ivy), a typical representative of the plant family Lamiaceae. Biochemical analyses indicated that the G. hederacea agglutinin (Gleheda) is a tetrameric protein consisting of four subunits pairwise linked through an interchain disulphide bridge and exhibits a preferential speci®city towards N-acetylgalactosamine. Cloning of the corresponding gene and molecular modeling of the deduced sequence demonstrated that Gleheda shares high sequence similarity with the legume lectins and exhibits the same overall fold and three-dimensional structure as the classical legume lectins. The identi®cation of a soluble and active legume lectin ortholog in G. hederacea not only indicates that the yet unclassi®ed Lamiaceae lectins belong to the same lectin family as the legume lectins, but also sheds a new light on the speci®city, physiological role and evolution of the classical legume lectins.
International Journal of Research and Analytical Reviews (IJRAR) , 2024
A protein fraction exhibiting lectin activity was identified in the extract of local Roman bean (Phaseolus vulgaris) sprouts. The galactose-specific lectins demonstrated the highest activity. The galactose-specific lectin (BS-Gal) was purified using affinity chromatography on an ES-2100-100 column. Gel electrophoresis revealed that BS-Gal consists of two protein subunits with molecular weights of 60 and 54 kDa, respectively. Various physicochemical properties of the lectin were investigated, showing optimal activity within a pH range of 7-9 and temperatures from 0 to 75 °C, beyond which its hemagglutinating activity diminishes. The lectin displayed high sensitivity to divalent ions and certain amino acids. Calcium and magnesium ions significantly contribute to the structural integrity of the lectin, as indicated by changes in hemagglutinating activity in the presence of EDTA and EGTA. Additionally, BS-Gal was found to bind to human erythrocytes expressing A and B antigens.
Identification and Characterization of Lectins from Leguminosae Plants
https://www.ijhsr.org/IJHSR\_Vol.9\_Issue.2\_Feb2019/IJHSR\_Abstract.017.html, 2019
Introduction: Since lectins are widely found in Leguminosae family due to their high protein content, in the present study, an attempt has been made to identify such legume lectins which have the agglutination properties with the red blood cells from normal individuals. Methods: Normal blood samples were collected, using the finger-prick technique. All blood samples were washed thrice in physiological saline and re-suspended at a concentration of 2% in normal saline. For ABO typing, standard serological procedure were followed. Results: In the results, various lectins showed the hemagglutination reaction pattern with human ABO blood groups. The lectin reacted with various blood groups with the strength of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128. Conclusion: The research findings were considered as the first step towards the field of sports nutrition. The lectin seeds are collected from the daily routine of the sports person, without knowing the effect of seeds on the body. Properly denaturized seeds should be consumed.
β-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.