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Pokeweed Antiviral Protein Project Yr3

Pokeweed Antiviral Protein Project Yr3. Supervisors: Simon Richardson and Paul Dyer By Amanda Aryee. Background. A plant containing type 1 ribosome inactivating protein (RIP) PAP (Pokeweed antiviral protein). It catalytically inactivates ribosome's, thus inhibiting protein synthesis.

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Pokeweed Antiviral Protein Project Yr3

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  1. Pokeweed Antiviral Protein Project Yr3 Supervisors: Simon Richardson and Paul Dyer By Amanda Aryee

  2. Background • A plant containing type 1 ribosome inactivating protein (RIP) PAP (Pokeweed antiviral protein). It catalytically inactivates ribosome's, thus inhibiting protein synthesis. • Depurination in ribosome's shown to occur in functionally equivalent positions in prokaryotes and eukaryotes. • Current drug delivery systems, although effective, have their advantages and disadvantages and can be improved.

  3. Methodology • Initially PAP will be sub cloned: recombinant PCR will be used to mutate the PAP sequence creating two combinations; • 1. Mutated (point mutation) R68G with active site intact. • 2. Mutated R68G with mutated active site. • Expression by bacterial expression cassette: pET151/D-TOPO • Evaluation of protein by Western Blotting: an analytical technique using antibody protein binding to detect specific proteins.

  4. A protein antibody-antibody sandwich is made. • Secondary antibody has horse radish peroxidase enzyme which converts a luminol substrate to a light releasing substance. Its intensity helps determine the volume of protein relative to other spots • From this it can be evaluated whether optimal expression and cloning has occurred Evaluations

  5. Likely Outcomes • Although there are drawbacks the hopeful outcomes are; • Improved PAP production in pET151/DTOPO when R68G is cloned. Optimal conditions obtained for two cloning types. • Modification of PAP sequence, for least cytotoxicity to cell of drug delivery. R68G mutation will enhance this. • The expected result is for the best possible sequence combination which allows for non damaging drug delivery. The second clone will be less cytotoxic thus more effective.

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