Patrícia M. G. Paiva

Protein Purification by Affinity Chromatography

Affinity chromatography is a method which depends essentially on the interaction between the molecule to be purified and a solid phase that will allow the separation of contaminants. Lectins are carbohydrate-binding proteins which can be purified by affinity chromatography; also, the presence of multiple molecular forms of lectins in a preparation can be separated. Immobilized lectins have been useful to affinity protein purification. In immunoaffinity chromatography an antibody or an antigen is immobilized on a support so as to purify the protein against which the antibody was developed. Monoclonal antibodies are extremely useful as immunosorbents for purification of antigen. Immobilization of monoclonal antibody on a suitable material to the column produces a support that will bind with high selectivity to protein against which the antibody was developed. Affinity chromatography containing DNA is a highly specific and important technique for the purification of DNA-binding proteins involved in the transcription, replication and recombination. The success of affinity chromatography depends on the conditions used in each chromatographic step. So, the optimization of protocol is essential to achieve optimal protein purification with maximum recovery.

Antimicrobial lectin from Schinus terebinthifolius leaf.

Schinus terebinthifolius leaves are used for treating human diseases caused by micro‐organisms. This work reports the isolation, characterization and antimicrobial activity of S. terebinthifolius leaf lectin (SteLL).

Water‐soluble Moringa oleifera lectin interferes with growth, survival and cell permeability of corrosive and pathogenic bacteria

This work evaluated the antibacterial activity of a water‐soluble Moringa oleifera seed lectin (WSMoL) by evaluating its effect on growth, survival and cell permeability of Bacillus sp., Bacillus cereus, Bacillus pumillus, Bacillus megaterium, Micrococcus sp., Pseudomonas sp., Pseudomonas fluorescens, Pseudomonas stutzeri and Serratia marcescens. In addition, the effect of lectin on membrane integrity of most sensitive species was also evaluated. All the tested bacteria are able to cause biocorrosion and some are also responsible for human infections.

Metal-sensitive and thermostable trypsin from the crevalle jack (Caranx hippos) pyloric caeca: purification and characterization

BackgroundOver the past decades, the economic development and world population growth has led to increased for food demand. Increasing the fish production is considered one of the alternatives to meet the increased food demand, but the processing of fish leads to by-products such as skin, bones and viscera, a source of environmental contamination. Fish viscera have been reported as an important source of digestive proteases with interesting characteristics for biotechnological processes. Thus, the aim of this study was to purify and to characterize a trypsin from the processing by-products of crevalle jack (Caranx hippos) fish.ResultsA 27.5 kDa trypsin with N-terminal amino acid sequence IVGGFECTPHVFAYQ was easily purified from the pyloric caeca of the crevalle jack. Its physicochemical and kinetic properties were evaluated using N-α-benzoyl-DL-arginine-p-nitroanilide (BApNA) as substrate. In addition, the effects of various metal ions and specific protease inhibitors on trypsin activity were determined. Optimum pH and temperature were 8.0 and 50°C, respectively. After incubation at 50°C for 30 min the enzyme lost only 20% of its activity. Km, kcat, and kcat/Km values using BApNA as substrate were 0.689 mM, 6.9 s-1, and 10 s-1 mM-1, respectively. High inhibition of trypsin activity was observed after incubation with Cd2+, Al3+, Zn2+, Cu2+, Pb2+, and Hg2+ at 1 mM, revealing high sensitivity of the enzyme to metal ions.ConclusionsExtraction of a thermostable trypsin from by-products of the fishery industry confirms the potential of these materials as an alternative source of these biomolecules. Furthermore, the results suggest that this trypsin-like enzyme presents interesting biotechnological properties for industrial applications.

Multi-effect of the water-soluble Moringa oleifera lectin against Serratia marcescens and Bacillus sp.: antibacterial, antibiofilm and anti-adhesive properties.

To evaluate the antibiofilm potential of water‐soluble Moringa oleifera seed lectin (WSMoL) on Serratia marcescens and Bacillus sp.

Selection of a protein solubilization method suitable for phytopathogenic bacteria: a proteomics approach.

BackgroundFinding the best extraction method of proteins from lysed cells is the key step for detection and identification in all proteomics applications. These are important to complement the knowledge about the mechanisms of interaction between plants and phytopathogens causing major economic losses. To develop an optimized extraction protocol, strains of Acidovorax citrulli, Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum were used as representative cells in the study of phytopathogenic bacteria. This study aims to compare four different protein extraction methods, including: Trizol, Phenol, Centrifugation and Lysis in order to determine which are more suitable for proteomic studies using as parameters the quantity and quality of extracted proteins observed in two-dimensional gels.ResultsThe bacteria studied showed different results among the tested methods. The Lysis method was more efficient for P. carotovorum subsp. carotovorum and R. solanacearum phytobacteria, as well as simple and fast, while for A. citrulli, the Centrifugation method was the best. This evaluation is based on results obtained in polyacrylamide gels that presented a greater abundance of spots and clearer and more consistent strips as detected by two-dimensional gels.ConclusionsThese results attest to the adequacy of these proteins extraction methods for proteomic studies.

Binding targets of termiticidal lectins from the bark and leaf of Myracrodruon urundeuva in the gut of Nasutitermes corniger workers: Binding targets of termiticidal lectins

BACKGROUND Lectins, carbohydrate-binding proteins, from the bark (MuBL) and leaf (MuLL) of Myracrodruon urundeuva are termiticidal agents against Nasutitermes corniger workers and have been shown to induce oxidative stress and cell death in the midgut of these insects. In this study, we investigated the binding targets of MuBL and MuLL in the gut of N. corniger workers by determining the effects of these lectins on the activity of digestive enzymes. In addition, we used mass spectrometry to identify peptides from gut proteins that adsorbed to MuBL-Sepharose and MuLL-Sepharose columns. RESULTS Exoglucanase activity was neutralized in the presence of MuBL and stimulated by MuLL. α-l-Arabinofuranosidase activity was not affected by MuBL but was inhibited by MuLL. Both lectins stimulated α-amylase activity and inhibited protease and trypsin-like activities. Peptides with homology to apolipophorin, trypsin-like enzyme, and ABC transporter substrate-binding protein were detected from proteins that adsorbed to MuBL-Sepharose, while peptides from proteins that bound to MuLL-Sepharose shared homology with apolipophorin. CONCLUSION This study revealed that digestive enzymes and transport proteins found in worker guts can be recognized by MuBL and MuLL. Thus, the mechanism of their termiticidal activity may involve changes in the digestion and absorption of nutrients.

Insecticide Activity of Lectins and Secondary Metabolites

Proteins are polymers of amino acids (molecules containing an amino group, a carboxylic group and a hydrophobic or hydrophilic side chain) present in all organisms. Apolar, polar uncharged and electrically charged amino acids are covalently linked through peptide bonds (amide bonds) and the sequence they form in the polypeptide chain (primary structure) determines the tertiary or quaternary structures ultimately presenting some biological activity. Proteins can be formed by one or multiple polypeptides (subunits) with or without a non-amino acid molecule (carbohydrate, ion, lipid, etc) linked to them. Lectins comprise a heterogeneous group of non-immune proteins that interact with carbohydrates. This interaction is behind a number of biological properties, including antimicrobial, antitumoral, hemagglutinating, mitogenic and insecticide activities. The specificity of the carbohydrate binding site is determined by the amino acids forming the lectin molecule, as well as shape and the spatial arrangement of neighboring amino acids; additionally, metal ions may contribute for correct positioning of the amino acid residues for binding to the carbohydrate (Sharon and Lis, 2001). Lectins can be divided into those that bind monosaccharides as well as oligosaccharides, and those that recognize only oligosaccharides (Sharon and Lis, 2007). Depending on carbohydrate specificity, they can be classified as: glucose/mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid-binding lectins (Wu et al., 2001). The hemagglutinating activity assay (Figure 1A) in presence of free carbohydrates (Figure 1B) has been proved to be a useful tool to characterize lectin specificity. Plant lectins have been isolated from bark, cladodes, flowers, leaves, rhizomes, roots and seeds. They differ from each other with respect to their molecular structures, carbohydratebinding specificities, and biological activities. The compact globular structures, molecular aggregation and glycosylation of lectins in general result in high structural stability (Kawsar et al., 2008; Moreno et al., 2008). In general, lectin isolation procedures include protein extraction steps with aqueous solvent, the production of a lectin-rich fraction, and separation of lectin from protein or non-protein