purcc 2012 - University of the Pacific

Poster Session Abstracts
synthesized for medical purposes more quickly
than by natural spider silk production. The
Pyriform Spidroin 1 (PySp1) protein of the black
widow spider, Latrodectus hesperus, contains an
AEQ rich region that participates in fibers that
cement dragline silk to solid supports. The
mRNA transcripts of PySp1 are expressed and
produced in the pyriform gland of the abdomen
of the spider. Since PySp1 transcripts are
expressed in the pyriform gland and the protein
spun into fibers, we hypothesize that PySp1 has
an important structural role as it is spun into
attachment disc silks to lock dragline fibers to
substrates, which is important for prey capture
and movement. To produce PySp1
recombinantly, the cDNA was inserted into the
prokaryotic expression vector pBAD-Thio-Topo.
PySp1 protein induction with arabinose was
checked with blue-silver staining and Western
blot analysis. The recombinant protein will be
purified using a nickel resin and then used to
spin synthetic fibers.
Expression of Internal Block Repeats
within the PySp2 Protein
Isaac Lee, Sally Ok, Thao Tran
Faculty Mentor: Craig Vierra
Spider silk is known for its high performance
mechanical properties. Spider silk is tougher
than high-tensile steel, largely due to the silk’s
increased extensibility. The key feature that
allows spider silk to possess phenomenal
mechanical properties is due to internal block
repeats within its protein architecture; these
repeats are rich in alanine and glycine. In
addition to the internal block repeats, spider silk
fibroins all share a non-repetitive N-terminus and
C-terminus, along with molecular masses that
exceed 250 kDa. Nephila clavipes, commonly
referred to as the golden orb weaving spider, has
6-7 silk glands. One of these glands, the
pyriform gland, expresses glue silk genes such as
PySp2. PySp2 is found in attachment discs and
facilitates N. clavipes in locomotion by helping
secure dragline silk to solid supports. Pyriform
silks are essential for a spider’s survival and this
material is spun into a liquid that dries quickly.
In an effort to analyze the relationship between
the mechanical properties and protein sequence
of PySp2, part of the PySp2 cDNA was
expressed in bacteria using the prokaryotic
expression vector pBAD/Thio-TOPO. The
recombinant protein was purified, and then
visualized using Coomassie blue staining as well
as western blot analysis. The long-term goal of
our research is to ultimately spin the purified
protein into fibers for mechanical testing.
Expression of Spider Egg Case Protein-3
for Structural and Mechanical Studies
Albert Lin
Faculty Mentor: Craig Vierra
Spiders produce a diverse number of silk
proteins that are well-known for their
extraordinary mechanical and biological
properties after they are spun into fibers.
Dragline silk has been the most prominent focus
of research because of its exceptional high
tensile strength and extensibility. In our research
we have focused on the characterization of a
third constituent of tubuliform silks (egg case
silk). This constituent, which is dubbed Egg
Case Protein 3 (ECP-3), is produced exclusively
in the tubuliform gland of the black widow
spider, Lactrodectus hesperus. The ECP-3
cDNA was obtained from a cDNA library
prepared from the silk-producing glands of the
spider. This cDNA was placed into the
prokaryotic expression vector pBAD-TOPO and
then moved into pET-19b-Sumo to facilitate
purification. Using western blot analysis, we
have verified that the ECP-3 protein is efficiently
expressed in bacteria. To test the structural
properties of ECP-3, we plan to perform circular
dichroism to study the secondary structure of
ECP-3. Our long-term goal is to spin artificial
silk fibers for mechanical studies to relate the
structure of ECP-3 to the material properties of
tubuliform silks.
Synthetic glue silk fibers spun from
PySp2 of orb-weaving spiders
Taylor Rabara, Aneesha Sharma, Reisa Rara
Faculty Mentor: Craig Vierra
Due to certain characteristics of spider silk, such
as high elasticity and tensile strength, researchers
are continuously expanding their knowledge of
spider silk proteins in the hopes of one day being
able to manufacture these silks for industrial
purposes. Some practical uses of spider silk
include bulletproof vests and medical sutures.
Ultimately, the goal is to be able to produce a
spider silk-like protein for artificial fiber
spinning.
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