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dc.contributor.advisor Ncube, I. Ngoepe, Mafora Gloria
dc.contributor.other Howard, R.L. 2013-05-28T12:08:25Z 2013-05-28T12:08:25Z 2011
dc.description Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2011 en_US
dc.description.abstract 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. en_US
dc.description.sponsorship 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 en_US
dc.format.extent xx, 118 leaves : col ill. en_US
dc.language.iso en en_US
dc.publisher University of Limpopo (Turfloop Campus) en_US
dc.relation.requires pdf en_US
dc.subject Mukwa (Pterocarpus angolensis) en_US
dc.subject Escherichia coli en_US
dc.subject Saccharomyces cerevisiae en_US
dc.subject Yarrowia lipolytica en_US
dc.subject Pal recombinant vector en_US
dc.subject Aspergillus niger en_US
dc.subject.ddc 572.60968 en_US
dc.subject.lcsh Proteins en_US
dc.subject.lcsh Plant metabolism -- South Africa en_US
dc.title Heterologous expression of a Mukwa (pterocarpus angolensis ) seed lectin (Pal) gene in Escherichia coli, Saccharomyces cerevisiae and Yarrowia lipolytica and construction of Pal recombinant vector for expression in Aspergillus niger en_US
dc.type Thesis en_US

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