Title

The Role of Calcium on Betagalactosidase Expression in Pichia pastoris

Poster Number

45

Lead Author Major

Biological Sciences

Format

Poster Presentation

Faculty Mentor Name

Der Thor

Faculty Mentor Department

Biological Sciences

Abstract/Artist Statement

Pichia pastoris is a yeast commonly used in heterologous protein expression due to its strong, inducible promoter, AOX1. Like many cells, P. pastoris is highly influenced by calcium, which acts as a messenger in signal transduction pathways and affects protein expression. EDTA is a polyamino carboxylic acid that chelates extracellular calcium molecules. The purpose of this experiment is to determine whether or not calcium affects protein expression in Pichia pastoris. In this experiment, Pichia pastoris was grown in yeast peptone dextrose. After an overnight growth, we transferred the overnight culture into four tubes of different environments: yeast nitrogen-based minimal medium (YNM), YNM plus CaCl2 (10mM), YNM plus EDTA (0.1mM), and YNM plus CaCl2 and EDTA (10mM and 0.1mM, respectively). Growth curves showed an increased growth with medium containing calcium and reduced growth in EDTA. Beta-galactosidase assays showed that EDTA increased the protein expression of the yeast, despite chelating of extracellular calcium. Based on the results found, we can conclude that blocking extracellular calcium using EDTA increases AOX1 driven expression of betagalactosidase.

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

The Role of Calcium on Betagalactosidase Expression in Pichia pastoris

DeRosa University Center, Ballroom

Pichia pastoris is a yeast commonly used in heterologous protein expression due to its strong, inducible promoter, AOX1. Like many cells, P. pastoris is highly influenced by calcium, which acts as a messenger in signal transduction pathways and affects protein expression. EDTA is a polyamino carboxylic acid that chelates extracellular calcium molecules. The purpose of this experiment is to determine whether or not calcium affects protein expression in Pichia pastoris. In this experiment, Pichia pastoris was grown in yeast peptone dextrose. After an overnight growth, we transferred the overnight culture into four tubes of different environments: yeast nitrogen-based minimal medium (YNM), YNM plus CaCl2 (10mM), YNM plus EDTA (0.1mM), and YNM plus CaCl2 and EDTA (10mM and 0.1mM, respectively). Growth curves showed an increased growth with medium containing calcium and reduced growth in EDTA. Beta-galactosidase assays showed that EDTA increased the protein expression of the yeast, despite chelating of extracellular calcium. Based on the results found, we can conclude that blocking extracellular calcium using EDTA increases AOX1 driven expression of betagalactosidase.