L. C. B. B. Coelho

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.

Mitogenic response and cytokine production induced by cramoll 1,4 lectin in splenocytes of inoculated mice.

Cramoll 1,4 is a lectin with specific glucose/mannose binding, which is extracted from seeds of Cratylia mollis Mart. Many assays have shown the cytokine expression activity and anti‐inflammatory profile of this lectin. The aim of this study was to evaluate the immunostimulatory response, in vitro, of splenocytes in mice previously inoculated, in vivo, with C. mollis (Cramoll 1,4) and Canavalia ensiformis (Con A) lectins. Results demonstrated higher proliferation indexes induced by Cramoll 1,4 than Con A lectin in relation to all experimental groups. Cramoll 1,4 and Con A also induced high levels of IL‐2, IL‐6, IFN‐γ and nitric oxide production. Moreover, Cramoll 1,4 did not induce apoptosis and stimulated a significant number of cells in the S phase of the cell cycle. Results showed that Cramoll 1,4 lectin induces proliferative response and suggested that this lectin can be used as a mitogenic agent in immunostimulatory assays.

Antimicrobial Activity of Cladonia verticillaris Lichen Preparations on Bacteria and Fungi of Medical Importance

Cladonia verticillaris lichen lectin (ClaveLL) was purified using a previously established protocol and then evaluated for its potential antimicrobial activity. Initially, the autochthonous lichen was submitted to extraction with sodium phosphate buffer pH 7.0, followed by filtration and centrifugation to obtain crude extract. A salt fractionation was performed with 30% ammonium sulfate. After centrifugation, the protein fraction was loaded onto molecular exclusion chromatography using Sephadex G-100 matrix to purify active lectin. ClaveLL showed antibacterial activity against Gram-positive (Bacillus subtilis, Staphylococcus aureus, and Enterococcus faecalis) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) assayed strains, with greater inhibitory effect on growth of E. coli (MIC of 7.18u2009μgu2009mL−1). The lowest minimum bactericidal concentration (MBC, 57.4u2009μgu2009mL−1) was detected against E. faecalis. The antifungal assay performed with Trichophyton mentagrophytes, Microsporum gypseum, Trichophyton rubrum, Trichosporon cutaneum, and Trichosporon asahi evaluated crude extract, fraction, and ClaveLL preparations. ClaveLL was the most active against T. rubrum with an inhibition percentage of 35% compared to negative control (phosphate buffer). Extract and fraction showed better activity on growth inhibition of T. mentagrophytes (35%). The results indicate the potential of ClaveLL and other C. verticillaris preparations as antimicrobial agents useful for applications focusing on human health.