This study has demonstrated and investigated the expression of a cDNA, coding for the pea seed storage protein vicilin, in the yeast, Saccharomyces cerevisiae. The cDNA was contained in the plasmid pLG1.63 and has been characterised and sequenced. The sequence showed that the cDNA coded for a 47KDa type of vicilin with a putative 24 amino-acid signal peptide, a proteolytic cleavage site and one glycosylation signal. The cDNA was cloned into two yeast expression vectors. The first utilised the GALIO promoter rendering expression of the cDNA inducible galactose, the construct was called pDUB2300. The second construct, pDUB2302, placed the cDNA under the control of the PGK promoter, rendering the cDNA constitutively expressed. When transformed into yeast, both constructs produced an immunoreactive vicilin species of M(_r) =49KDa. In the case of pDUB2302 the protein was produced at up to 5.5% of total cell protein. The protein was shown to be associated with a particulate fraction and displayed altered precipitation characteristics when compared with pea vicilin. By using tunicanydn and N-glycosidase, the protein was shown to be unglycosylated. Partial purification and (^35)S-methionine labelling demonstrated that the signal pep tide remained uncleaved. Cell fractionation studies indicated that vicilin was enriched in the yeast microsomal fraction, suggesting that vicilin was located in the EH. This was confirmed electron microscopy of immuno-gold labelled yeast which showed vicilin associated with the ER. The electron micrographs also suggested that a small proportion of the protein might be reaching thecolgi apparatus and the vacuole membrane. The presence of specific cleavage products on some western blots suggested that vicilin possessed a cleavage site for a yeast protease, though whether this was the same site as the pea proteolytic cleavage site was not determined. The pattern and nature of the expression of vicilin from this cDNA was discussed in the context of heterologous protein expression in yeast in general and plant storage protein expression in yeast in particular.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:237532 |
| Date | January 1989 |
| Creators | Stewart, Gregor James |
| Publisher | Durham University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.dur.ac.uk/9350/ |
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