A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6-7 and its highest measured initial activity at 100 degrees C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 degrees C.
The thermostable cellulase Cel12A from Rhodothermus marinus was produced at extremely low levels when expressed in Escherichia coli and was cytotoxic to the cells. In addition, severe aggregation occurred when moderately high concentrations of the enzyme were heat-treated at 65 degrees C, the growth optimum of R. marinus. Sequence analysis revealed that the catalytic module of this enzyme is preceded by a typical linker sequence and a highly hydrophobic putative signal peptide. Two deletion mutants lacking this hydrophobic region were cloned and successfully expressed in E. coli. These results indicated that the N-terminal putative signal peptide was responsible for the toxicity of the full-length enzyme in the host organism. This was further corroborated by cloning and expressing the hydrophobic N-terminal domain in E. coli, which resulted in extensive cell lysis. The deletion mutants, made up of either the catalytic module of Cel12A or the catalytic module and the putative linker sequence, were characterised and their properties compared to those of the full-length enzyme. The specific activity of the mutants was approximately three-fold higher than that of the full-length enzyme. Both mutant proteins were highly thermostable, with half-lives exceeding 2 h at 90 degrees C and unfolding temperatures up to 103 degrees C.
CD8+ Tregs display an immunoregulatory activity and may play an essential role in the immunopathology of several diseases. Therefore, their therapeutic potential is exquisite and further studies on their differentiation and function are essential. The aim of this study was to evaluate the role of the innate immune system in CD8 + iTreg differentiation and function. Naive human CD8 + CD25 À CD45RA + T cells were cultured in Treg-inducing conditions with or without IL-1b, TNFa or monocyte-derived dendritic cells (DCs). The differentiation of CD8 + CD127 À CD25 hi FoxP3 hi -induced Tregs (CD8 + iTregs) is dependent on TGF-b1 and IL-2, which had synergistic effect upon their differentiation. CD8 + iTregs were also induced in a coculture with allogeneic mature DCs (mDCs). The CD8 + iTregs suppressive function was confirmed, which was diminished in the presence of IL-1b and TNFa. The IL-1b-prevented suppressive function was associated with reduced secretion of IL-10 and IFNc, whereas the presence of TNFa did not affect their secretion. Furthermore, the presence of TNFa reduced IL-10 and TGF-b1 secretion by CD8 + iTregs, whereas only IL-10 secretion was decreased by IL-1b. Together, these results suggest that IL-1b and TNFa prevent IL-2-and TGF-b1-driven CD8 + iTregs suppressive function in human T cells. Such pro-inflammatory innate immune response possibly mediates its negative tolerogenic effect through reduced IFNc-, IL-10-and TGF-b1-driven mechanism.
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