Expression and Purification of the Pyriform Spidroin 2 Protein of Orb-Weaving Spiders in Pichia pastoris
Poster Number
43
Format
Poster Presentation
Faculty Mentor Name
Geoffrey Lin-Cereghino
Faculty Mentor Department
Biological Sciences
Additional Faculty Mentor Name
Joan Lin-Cereghino
Abstract/Artist Statement
The high extensibility and toughness of spider silk make it an ideal material for various industrial uses. Mass production of spider silk, however, remains an obstacle. One possible method is to express spider silk protein in Pichia pastoris, a methylotrophic yeast. This yeast can be grown in high concentrations, is easily genetically manipulated, can perform numerous eukaryotic posttranslational modifications, and has been frequently used to express large amounts of foreign proteins. The goal of this project was to express the Pyriform Spidroin 2 Protein (PySp2), an attachment disk glue silk protein found in orb-weaving spiders, in Pichia pastoris and then to purify the expressed protein. After growth and induction of PySp2, expression of the protein was confirmed through western analysis and optimized by varying culture conditions. PySp2 was then purified from extracellular supernatants using affinity chromatography in native and denaturing conditions. Addition of the denaturant guanidine hydrochloride to supernatant prior to affinity chromatography yielded the most effective purification conditions. Ultimately, the properties of silk fibers spun from heterologously expressed PySp2 protein will be compared to naturally produced spider silk fibers. This will help determine whether Pichia pastoris is an ideal resource to synthesize spider silk proteins on a larger scale.
Location
DeRosa University Center, Ballroom
Start Date
20-4-2013 1:00 PM
End Date
20-4-2013 3:00 PM
Expression and Purification of the Pyriform Spidroin 2 Protein of Orb-Weaving Spiders in Pichia pastoris
DeRosa University Center, Ballroom
The high extensibility and toughness of spider silk make it an ideal material for various industrial uses. Mass production of spider silk, however, remains an obstacle. One possible method is to express spider silk protein in Pichia pastoris, a methylotrophic yeast. This yeast can be grown in high concentrations, is easily genetically manipulated, can perform numerous eukaryotic posttranslational modifications, and has been frequently used to express large amounts of foreign proteins. The goal of this project was to express the Pyriform Spidroin 2 Protein (PySp2), an attachment disk glue silk protein found in orb-weaving spiders, in Pichia pastoris and then to purify the expressed protein. After growth and induction of PySp2, expression of the protein was confirmed through western analysis and optimized by varying culture conditions. PySp2 was then purified from extracellular supernatants using affinity chromatography in native and denaturing conditions. Addition of the denaturant guanidine hydrochloride to supernatant prior to affinity chromatography yielded the most effective purification conditions. Ultimately, the properties of silk fibers spun from heterologously expressed PySp2 protein will be compared to naturally produced spider silk fibers. This will help determine whether Pichia pastoris is an ideal resource to synthesize spider silk proteins on a larger scale.