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.
 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.
Methodology
 Initially PAP will be sub cloned: recombinant PCR will
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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/DTOPO
Evaluation of protein by Western Blotting: an
analytical technique using antibody protein binding to
detect specific proteins.
Evaluations
•A protein antibodyantibody 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
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.