One particular mechanism of resistance to organophosphate and carbamate insecticides is insensitive variants of the target enzyme acetylcholinesterase (AChE). The aims were to set up an expression system in Saccharomyces cerevisiae for insect AChE with the long term objective of studying mosquito AChE variants and to assess the potential of a yeast system for the large scale production of AChE. Initially work centred on the analysis of an expression construct pBM150-DAChE containing the Drosophila melanogaster AChE (DAChE) cDNA under the yeast GAL10 promoter. Preliminary analyses were conducted using the assay developed by Ellman et al (1961). High levels of interference were encountered on cell breakage making assessments of expression extremely difficult. This interference was found to be due to thiol groups within the yeast cell wall. Enzymatic digestion of the cell wall was found to reduce interference to a manageable level. Analysis of protoplasts indicated that active AChE was either not being expressed or being expressed at undetectable levels. AChE mRNA could not be detected by Northern blotting. The DAChE cDNA was ligated into the high copy number vector pG3 under the constitutive glyceraldehyde-3-phosphate dehydrogenase gene (GPD) promoter to form the construct pG3-DAChE which was transformed into the BJ2168 strain of S. cerevisiae. Attempts were also made to subclone the DAChE cDNA into a steroid inducible vector (p2UG) and a secretion vector (pPIC9). Analysis of expression from construct pG3-DAChE by both the Sabine (1955) and Ellman methods revealed that biologically active AChE was being constitutively expressed by S. cerevisiae. The expressed DAME was further authenticated by inhibition with the insecticide Bendiocarb and the inhibitor phenylmethylsulfonyl fluoride. A Kc. t value f or the expressed DAChE was approximated at 6429.3 molecules s-1 but specific activities were found to be low (--0.05 Units). The percentage of total S. cerevisiae protein that was active DAME was estimated at 0.0009%. SDS-PAGE analysis indicated that this active percentage was likely to be commensurate with the total amount of enzyme protein translated. Northern blotting of total RNA detected low levels of a shortened transcript that may either translate to a truncated but enzymatically active DAME or may represent a transiently stabilised mRNA in the process of degradation. The presence of a small population of full length transcripts that remained undetected should not be discounted. The expressed DAChE was found to be located in the cell membrane of s. cerevisiae suggesting that it had passed through the secretory pathway and further that the active site was probably internal of the membrane rather than externally situated. Plasmid copy number estimations and growth rate analyses showed that the expressed DAChE did not have a deleterious effect on cellular metabolism. RT-PCR was used to generate a homologous cDNA probe that could in the future be used to screen a cDNA library from Culex molestus for a susceptible mosquito AChE gene.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:391819 |
| Date | January 1998 |
| Creators | Stopps, Kevin Cyril Andrew |
| Publisher | Queen Mary, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://qmro.qmul.ac.uk/xmlui/handle/123456789/28948 |
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