The Role of Calcium on Betagalactosidase Expression in Pichia pastoris
Poster Number
45
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
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.