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

by Rochereau, Nicolas

Abstract (Summary)
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-?-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-?-glucosidase in rye. The aim was to determine the properties of a cell wall ?-glucosidase. Two different systems for expression and purification of ?-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the ?-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 ?-glucosidase solubility was determined. Finally a purification of the ?-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant ?-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-?-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 ?-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.
Bibliographical Information:

Advisor:

School:Södertörns högskola

School Location:Sweden

Source Type:Master's Thesis

Keywords:rye defense system ? glucosidase subcloning his tag thioredoxin e coli ni nta western blot

ISBN:

Date of Publication:08/09/2007

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