One-step purification of chimeric green fluorescent protein providing metal-binding avidity and protease recognition sequence.

Gene fusion technique was successfully applied as a potential approach to create a metal-binding site to assist one-step purification of green fluorescent protein (GFP). The chimeric GFP carrying hexapolyhistidine (H6GFPuv) was purified to homogeneous protein via the Immobilized Metal Affinity Chromatography charged with zinc ions. Removal of metal tagger could readily be performed by using enterokinase enzyme. Engineering of the hexahistidine and enterokinase cleavage sites (DDDDK) onto the chimeric protein did not significantly affect the fluorescent property and the binding avidity to Burkholderia pseudomallei protease of a chimeric protease-binding GFP (H6PBGFPuv). This concludes that engineering of repetitive histidine regions onto interested target protein along with the enterokinase cleavage sites will ease the complication of protein purification.

Specific binding of Burkholderia pseudomallei cells and their cell-surface acid phosphatase to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2).

Specific binding between bacterial cells and host tissue is an early step of the pathogenesis of infection. Burkholderia pseudomallei cells, the causative micro-organisms of melioidosis, were demonstrated to bind specifically to tissue glycolipids (asialo GM1 and asialo GM2) by solid phase binding assay on thin layer chromatograms. The detection limit was around 400 pmol of the glycolipids. Acid phosphatase purified from the culture filtrate of B. pseudomallei was tested for such binding properties, and the same results were obtained. According to our previous studies, the enzyme is a glycoprotein located on the cell surface, and hydrolysed tyrosine phosphate most actively among the substrates so far tested. The mode of binding between the enzyme and the glycolipids was analyzed by comparison of binding levels among three samples different in protein content, sugar content and specific phosphatase activities per protein and sugar residue. The results suggested the possibility of a receptor-ligand relationship between the bacterial enzyme and the host-cell glycolipids (asialo GM).

Affinity and response of Burkholderia pseudomallei and Burkholderia cepacia to insulin.

The cells of Burkholderia pseudomallei, B. cepacia and Pseudomonas aeruginosa grown on agar plates were stained with fluorescently-labeled insulin. The former two species were stained positively indicating insulin binding but P. aeruginosa was not. Insulin exposure reduced phospholipase C and acid phosphatase activities of B. pseudomallei but did not affect those enzymatic activities of B. cepacia in the employed experimental conditions. It is suggested that B. pseudomallei have insulin receptors which may be associated with a signal transfer system involving phospholipase and protein tyrosine phosphatase.

Evolution of cell-surface acid phosphatase of Burkholderia pseudomallei.

Acid phosphatase active fractions were obtained from cell-free extract, outermembrane fraction and culture filtrate of Burkholderia pseudomallei by column chromatography with sepharose 6B and DEAE cellulose. The comparison of the elution patterns of protein, sugar and enzymatic activity among these three components suggested that the enzyme is a glycoprotein evolving from premature proteins through glycosylation and that the enzyme is translocated during glycosylation from the cytoplasm to the outer membrane and finally excreted into the environment. When tunicamycin, a glycosylation inhibitor, was added to the culture, the peaks of sugar and enzymatic activity were lowered concomitantly leaving the protein peak unchanged in the elution pattern of the culture filtrate. The affinity of the bacterial surface to antienzyme sera was demonstrated by immuno-fluorescence microscopy.

Separation of antigenic glycoprotein fractions from cell-free homogenate of Pseudomonas pseudomallei and characterization as tyrosine phosphatase.

Cell-free extracts were prepared from Pseudomonas pseudomallei cells by freezing-thawing, sonication, and differential ultracentrifugation. The extracts were subjected to column chromatography with DEAE-sepharose to obtain glycoprotein fractions. The fractions showed acid phosphatase activity to p-nitrophenyl phosphate, tyrosine phosphate, serine phosphate, but not to threonine phosphate. They were highly antigenic when tested by immunofluorescence assay with the sera of melioidosis patients.

Effects of tunicamycin on the pH-activity pattern of acid phosphatase in Pseudomonas pseudomallei.

The liquid culture of Pseudomonas pseudomallei shows a complex feature in in the pH-activity pattern of acid phosphatase, not a single peak curve. There was an evident tendency that the higher activity shifted to the higher pH range with the growth of culture. The culture in the presence of tunicamycin (20 micrograms/ml) showed a decreased activity selectively in the higher pH range, while the activity in the lower pH was more heat-labile. The bacterial cells grown on agar plates containing tunicamycin were more heat-labile than the untreated control cells. The glucosidase-treatment reduced the enzymatic activity (of the phosphatase-active fractions from the living cells) with the shift of the optimum pH to lower pH. These observations together with some collateral findings suggest that the pH-activity pattern of acid phosphatase in P. pseudomallei is associated with the development of precursor enzyme proteins to mature glycoproteins.