The Structural Characterization of the Saccharomyces cerevisiae MATα Secretion Signal
Poster Number
25
Format
Poster Presentation
Faculty Mentor Name
Liang Xue
Faculty Mentor Department
Chemistry and Biological Sciences
Additional Faculty Mentor Name
Geoff Lin-Cereghino
Additional Faculty Mentor Name
Joan Lin-Cereghino
Abstract/Artist Statement
Other Contributors: Peter Wei, Sabreen Chahal The MATα prepro secretion signal, which consists of pre-region (amino acids 1-19) and pro-region (amino acids 20-85), is the most successful and commonly used secretion signal leader in the methylotrophic yeast Pichia pastoris. The yeast has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The MATα leader helps provide a high level of secretion to most proteins. However, limitations exist as some proteins cannot be secreted efficiently even with the MATα prepro secretion signal. Some strategies to enhance secretion efficiency involve modifying the secretion signal leader. Among various mutant types of MATα proteins modified by adding or deleting residues in the pre or pro region, the MATα with removal of 4 amino acids 57-60, known as MATα Δ57- 60, showed a 60% drop in secretion level. To physically analyze the actual secondary structure of the MATα Δ57-60, the mutant MATα was initially cloned by PCR, ligated into a pET SUMO vector, and expressed in BL21 E.coli. After small scale expression to optimize conditions for large scale expression, the MATα Δ57-60 was purified by histidine column affinity, dialysis, and SUMO digestion. The purified mutant MATα’s secondary structure was then determined by circular dichroism and compared to that of wild type MATα. By creating mutant MATα prepro signal leaders and analyzing their physical properties, we hope to better understand the signal leader’s structural properties in order to further increase secretion levels of heterologous proteins in P. pastoris.
Location
DeRosa University Center, Ballroom
Start Date
25-4-2015 2:00 PM
End Date
25-4-2015 4:00 PM
The Structural Characterization of the Saccharomyces cerevisiae MATα Secretion Signal
DeRosa University Center, Ballroom
Other Contributors: Peter Wei, Sabreen Chahal The MATα prepro secretion signal, which consists of pre-region (amino acids 1-19) and pro-region (amino acids 20-85), is the most successful and commonly used secretion signal leader in the methylotrophic yeast Pichia pastoris. The yeast has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The MATα leader helps provide a high level of secretion to most proteins. However, limitations exist as some proteins cannot be secreted efficiently even with the MATα prepro secretion signal. Some strategies to enhance secretion efficiency involve modifying the secretion signal leader. Among various mutant types of MATα proteins modified by adding or deleting residues in the pre or pro region, the MATα with removal of 4 amino acids 57-60, known as MATα Δ57- 60, showed a 60% drop in secretion level. To physically analyze the actual secondary structure of the MATα Δ57-60, the mutant MATα was initially cloned by PCR, ligated into a pET SUMO vector, and expressed in BL21 E.coli. After small scale expression to optimize conditions for large scale expression, the MATα Δ57-60 was purified by histidine column affinity, dialysis, and SUMO digestion. The purified mutant MATα’s secondary structure was then determined by circular dichroism and compared to that of wild type MATα. By creating mutant MATα prepro signal leaders and analyzing their physical properties, we hope to better understand the signal leader’s structural properties in order to further increase secretion levels of heterologous proteins in P. pastoris.