Expression and Purification of Pyriform Spidroin 2 Protein
Poster Number
60
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
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.