purcc 2012 - University of the Pacific

Poster Session Abstracts
contains a complex mixture of both positive and
negative cis-acting elements, suggesting that the
construction of a synthetic 5'UTR optimized for
a higher level of expression may be challenging.
Expression and Purification of Pyriform
Spidroin 2 Protein
Nadia Shaheen
Faculty Mentors: Joan Lin-Cereghino,
Geoff Lin-Cereghino
Pichia pastoris is a yeast commonly used for
expression of foreign proteins, as the yeast are
easily genetically manipulated, can be grown in
high concentrations, and express large amounts
of heterologous proteins. In this case, Pichia
pastoris was used to express the Pyriform
Spidroin 2 Protein, PySp2, a spider silk
attachment disk glue protein. After growth and
induction of PySp2, expression of the protein
was confirmed through western analysis.
Expression was optimized by varying culture
conditions. PySp2 was then purified from the
cultures via affinity chromatography using both
native and denaturing conditions. The protein
was successfully expressed on small and large
scales; however, purification in native conditions
resulted in a low yield. The yield from
denaturing conditions, on the other hand, was
significantly higher. Ultimately, the properties
of heterologously expressed PySp2 protein can
be compared to naturally produced PySp2
protein. This will help determine whether Pichia
pastoris is an ideal resource to synthesize spider
silk proteins on a larger scale.
The Structural Studies of Artificial Silk
Fibers and the PySp2 Protein
Hasan AlKazemi, Jacky Aguilar, Sophia
Chou
Faculty Mentor: Craig Vierra
Spider silk is a biodegradable, non-toxic
biopolymer that is stronger than Kelvar, Nylon,
and steel. Spider silk can be used in a variety of
fields, including engineering and medicine. In
our research, we are attempting to spin synthetic
spider silk from a glue silk protein, PySp2.
PySp2, which is expressed in golden orb
weavers, is spun into attachment discs and helps
immobilize dragline threads. PySp2 contains
internal block repeats whose sequences can be
tested for their unique mechanical properties.
Using genetic engineering, we inserted a
segment of the PySp2 cDNA into the prokaryotic
expression vector pBAD-Thio-TOPO.
Restriction digestion analysis and agarose gel
electrophoresis was performed to verify the
presence and directionality of the PySp2 cDNA
in the cloning vector. Following the
confirmation of the cDNA insert in the cloning
vector, we induced the expression of PySp2 in
bacteria and monitored its expression using
western blot analysis. Our long term goal is to
purify the PySp2 protein and spin artificial silk
fibers as well as use the solubilized protein for
structural studies.
Expression of the Latrodectus hesperus
Glue Silk Protein, Pyriform Spidroin 1, in
Bacteria
Richard Chen, Alex Hoang-Mendoza, Jun
Park, Pauline Pham, Moe Thien
Faculty Mentor: Craig Vierra
The biological mechanisms that spiders use to
spin silk fibers remains a mystery to scientists,
making it difficult from labs to biomimic this
process. Spiders spin multiple silk types that
have a diverse range of biological functions. The
highly studied dragline silk has been shown to
have tremendous properties that rival both steel
and Kevlar in elasticity, toughness, and tensile
strength. In this lab, the pyriform spidroin
protein 1 (PySp1) from the black widow spider,
Latrodectus Hesperus, was studied to help
elucidate how it provides a strong, adhesive
glue-like function that anchors dragline silk. The
purpose of our research was to express a portion
of the PySp1 protein. PCR was used to amplify
a segment of the PySp1 cDNA, which was then
inserted into the pBAD-Thio/TOPO bacterial
expression vector. E. coli was then transformed
with the ligation mixture and colonies carrying
the correct vector were identifies by restriction
digestion and agarose gel electrophoresis.
Transformants carrying the vector were induced
with arabinose to express the PySp1 cDNA
insert. Western blot analysis was then used to
check for the expression of the PySp1 protein.
With the results obtained, further experiments
will be carries out in attempt to synthesize
synthetic pyriform fibers.
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