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Rye cell wall β-glucosidase: subcloning, expression and purification of recombinant protein from E.coli
Södertörn University College, School of Life Sciences.
2007 (English)Independent thesis Basic level (professional degree), 20 points / 30 hpStudent thesis
Abstract [en]

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-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-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 β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-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 β-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.

Place, publisher, year, edition, pages
Huddinge: Institutionen för livsvetenskaper , 2007. , 21 p.
Keyword [en]
rye, defense system, β-glucosidase, subcloning, his-tag, thioredoxin-tag, E.coli, Ni-NTA, western blot
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:sh:diva-1329OAI: oai:DiVA.org:sh-1329DiVA: diva2:15397
Uppsok
bio-/geovetenskap
Supervisors
Examiners
Available from: 2007-08-09 Created: 2007-08-09

Open Access in DiVA

fulltext(345 kB)594 downloads
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6a289d582b566d50b5d553a20bca5e79737d486f3f0ef403b54a8a5a4e820bf53a72399d
Type fulltextMimetype application/pdf

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CiteExportLink to record
Permanent link

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Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf