Title

Expression and Purification of Pyriform Spidroin 2 Protein

Poster Number

60

Lead Author Major

Biological Sciences

Format

Poster Presentation

Faculty Mentor Name

Geoff Lin-Cereghino

Faculty Mentor Department

Biological Sciences

Additional Faculty Mentor Name

Joan Lin-Cereghino

Abstract/Artist Statement

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.

Location

Grave Covell

Start Date

21-4-2012 10:00 AM

End Date

21-4-2012 12:00 PM

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Apr 21st, 10:00 AM Apr 21st, 12:00 PM

Expression and Purification of Pyriform Spidroin 2 Protein

Grave Covell

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.