ABSTRACT
Objective To evaluate the effectiveness of ultrafiltration technology in endotoxin removal from purified recombinant MUC1-MBP fusion protein (MUC1-MBP)and to demonstrate the effect of ultrafiltration on endotoxin removal.Methods CM Sepharose FF weak cation exchange (CM)(CM group), CM combined with Phenyl Sepharose 6 FF exchange (C6)(CM+C6 group),CM combined with ultrafiltration (CM+ultrafiltration group), and CM combined with C6 and ultrafiltration (CM+C6+ultrafiltration group)were used to purify the MUC1-MBP from E.coli. and remove endotoxin;the expression level of endotoxin was detected by Chromogenic End-point Tachypleus Amebocyte Lysate.Results There was a single band at the expected molecular weight of 62 000 by SDS-PAGE analysis.and the purity>96% by Quantity One analysis.The endotoxin levels in CM group and CM +C6 group were quite high and there was no significant difference between two groups (P>0.05 );the endotoxin level in CM+ultrafiltration group was significantly lower than that in CM group, and there was significant difference (P0.05 ). Conclusion The effects of CM or CM combined with C6 on endotoxin removal are quite poor, especially C6;CM combined with ultrafiltration are quite effective on endotoxin removal,and ultrafiltration plays an important role in endotoxin removal.
ABSTRACT
BACKGROUND: Stimulation of Toll-like receptors (TLRs) by microbial products has been utilised to potentiate immune responses against haematologic malignancies. The maltose-binding protein (MBP) of Escherichia coli could induce the activation of immune cells via TLR4. The aim of the present study was to investigate whether TLRs mediated the biological effects of MBP on U937 and Jurkat cells in vitro. METHODS We observed the effect of MBP on U937 and Jurkat cells by using the WST, cell cycle analysis and morphological observation. Further, cells were stimulated with MBP for indicated times and doses, and detected by RT-PCR, western blotting, immunohistochemistry and immunofluorescence staining to investigate the mechanisms involved in cell viability. RESULTS: MBP enhanced the viability of U937 and Jurkat cells, and the effects were blocked by anti-TLR2, but not anti-TLR4 in U937 cells. Further studies confirmed that MBP was able to directly bind to U937 and Jurkat cells and modulate TLR expression. The effects of MBP depended on the activation of NF-κB and MAP kinase in U937 and Jurkat cells. CONCLUSIONS: Our results demonstrated that MBP could directly promote U937 cell viability via TLR2. It suggested that MBP may be used as an adjuvant for participating in the immunotherapy of haematologic malignancies.
