Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2011 / Pterocarpus angolensis seed lectin (PAL), a 28 kDa non glycosylated protein, was initially successfully cloned and expressed in E. coli for ease of high protein production. It was discovered, however, as in similar studies that the recombinant PAL yield in E. coli is low and localized intracellularly. This makes extraction even more difficult because most of the protein is lost either when the cell undergoes lysing or when there is incomplete extraction. As a result of the low yields in E. coli, expression vectors were constructed for pal expression in S. cerevisiae, Y. lipolytica and A. niger. Colony PCR of S. cerevisiae transformants confirmed the presence of pal gene whilst sequencing revealed a 66% homology to native PAL. Expression of recombinant PAL in S. cerevisiae, which was expected to be intracellular, was doubtfully unsuccessful since no signal was detected following Western blot analysis. A pBARMTE1-pal expression vector was successfully constructed and could be used for expression studies in Aspergillus niger, however, it was not used in this study. A pal gene whose codons were optimized for Y. lipolytica was synthesized and successfully cloned and expressed in Y. lipolytica. Gene sequence alignment of native pal and the codon optimized pal showed 81% homology whilst the amino acid alignment showed 100% homology. A 31 kDa, recombinant PAL was successfully expressed in Y. lipolytica. The recombinant PAL was approximately 3 kDa larger than native PAL. It was established that this is due to glycosylation of the recombinant PAL. This recombinant protein was found to be more thermostable than native PAL since it demonstrated haemagglutination activity after 10 minutes of exposure in a boiling water bath and only lost activity after 2 hours of exposure to boiling. This study succeeded in producing a more stable extracellular recombinant PAL which demonstrated biochemical activity that was largely similar to that of native PAL but only differed in carbohydrate specificity and haemagglutinating strengths. / Flemish Interuniversity Council (VLIR-UOS)-Own Initiative Project,the SARBIO- South African Regional Co-operation in Biochemistry, Molecular Biology and Biotechnology, the (CSIR) Council for Scientific and Industrial Research,the (NRF) National Research Foundation,(TBI) The Biovac Institute Foundation, and the (SIDA) Swedish International Agency
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ul/oai:ulspace.ul.ac.za:10386/857 |
| Date | January 2011 |
| Creators | Ngoepe, Mafora Gloria |
| Contributors | Ncube, I., Howard, R.L. |
| Publisher | University of Limpopo (Turfloop Campus) |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | xx, 118 leaves : col ill. |
| Relation |
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