Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase ( lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His6-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65 degrees C, respectively, and 50% of its activity remained after incubation at 60 degrees C for 32 min. The enzyme preferentially hydrolyzed L-leucine- p-nitroanilide ( L-Leu- p-NA) followed by Cys derivative.
Tournefortia sarmentosa, a Chinese herbal medicine, is considered an antioxidant or a detoxicating agent. Recently T. sarmentosa has received attention for its effects on the immune response. Here we provide evidence that aqueous extract of T. sarmentosa can induce increased phagocytic uptake of Escherichia coli by differentiated HL-60 cells and by neutrophils. Our results also revealed that T. sarmentosa can inhibit E. coli survival within differentiated HL-60 cells. Furthermore, aqueous extract of T. sarmentosa has been shown to enhance cell surface Mac-1 expression and the activated AKT signaling pathway in E. coli-stimulated neutrophils. We also examined the effect of each constituents in aqueous extract of T. sarmentosa on phagocytic uptake of E. coli by differentiated HL-60 cells or neutrophils. Bacterial survival, cell surface Mac-1 expression, and AKT activation of neutrophils were also examined. Our results showed that caffeic acid is an important constituent in mediating aqueous extract of T. sarmentosa-induced phagocytic uptake. Taken together, these results suggest that aqueous extract of T. sarmentosa exerts effects that enhance inflammatory responses by improving phagocytic capability, inhibiting bacterial survival within cells, and increasing Mac-1 expression of neutrophils.
The full-length Escherichia coli gamma -glutamyltranspeptidase (EcGGT) gene and five truncations lacking 33, 51, 54, 60, and 78 bp respectively at the 5' end were prepared by polymerase chain reaction and cloned into the expression vector pQE-30. Isopropyl-beta -D-thiogalactopyranoside induction of E. coli M15 cells bearing the recombinant plasmids resulted in the intracellular production of the expressed proteins, EcGGT, EcGGT/DeltaN11, EcGGT/DeltaN17, EcGGT/DeltaN18, EcGGT/DeltaN20, and EcGGT/DeltaN26. The overexpressed enzymes were purified to near homogeneity by Ni(2+)-NTA resin. The specific activity for EcGGT, EcGGT/DeltaN11 and EcGGT/DeltaN17 was 5.3, 4.9, and 4.8 U/mg protein respectively, whereas the rest three enzymes had shown no GGT activity under the enzyme assay conditions. More than 94% of the activity was found in the cytoplasmic fraction of E. coli M15 cells harboring pQE-EcGGT, pQE-EcGGT/DeltaN11 or pQE-EcGGT/DeltaN17. Western blot analysis confirmed that the majority of N-terminally truncated enzymes were present in the cytoplasm.
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