Rye cell wall ß-glucosidase: subcloning, expression and purification of recombinant protein from E.coli

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Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-ß-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-ß-glucosidase in rye. The aim was to determine the properties of a cell wall ß-glucosidase. Two different systems for expression and purification of ß-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the ß-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the ß-glucosidase solubility was determined. Finally a purification of the ß-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant ß-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-ß-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable ß-glucosidase activity. It was not possible to purify active enzyme. Fro...