A mannose-binding lectin, termed champedak lectin-M, was isolated from an extract of the crude seeds of champedak (Artocarpus integer). On gel filtration chromatography, the lectin eluted in a single peak at elution volumes corresponding to 64 kDa. SDS-PAGE showed the mannose-binding lectin to be composed of 16.8 kDa polypeptides with some of the polypeptides being disulphide-linked to give dimers. When tested with all isotypes of immunoglobulins, champedak lectin-M demonstrated a selective strong interaction with human IgE and IgM, and a weak interaction with IgA2. The binding interactions of lectin-M were metal ion independent. The lectin was also shown to interact with horseradish peroxidase, ovalbumin, porcine thyroglobulin, human alpha1-acid glycoprotein, transferrin and alpha1-antitrypsin. It demonstrated a binding preference to Man alpha 1-3Man ligands in comparison to Man alpha 1-6Man or Man alpha 1-2Man.
Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.
The effect of the mannose-binding champedak (Artocarpus integer) lectin-M on the cellular proliferation of murine lymphocytes was investigated in this study. Our data demonstrated that the lectin was the main mitogenic component in the crude extract of the champedak seeds. It stimulated the proliferation of murine T cells at an optimal concentration of 2.5 microg/ml in a 3 day culture. Lectin-M appeared to be a T-cell mitogen as it does not induce significant DNA synthesis when cultured with spleen cells from the nude mouse. In the absence of T cells, the lectin was incapable of inducing resting B cells to differentiate into immunoglobulin secreting plasma cells.
Helicobacter pylori is a human pathogen and during the process of infection, antigens from the bacterium elicit strong host humoral immune responses. In our previous report, native H. pylori UreG protein showed good reactivity with sera from H. pylori patients. This study was aimed at producing the recombinant form of the protein (rUreG) and determining its seroreactivities.