Title

Expression and Purification of the Pyriform Spidroin 2 Protein of Orb-Weaving Spiders in Pichia pastoris

Poster Number

43

Lead Author Major

Biological Sciences

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

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Apr 20th, 1:00 PM Apr 20th, 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.