Title

Expression and secretion of prochymosin in Pichia pastoris.

Poster Number

7

Format

Poster Presentation

Abstract/Artist Statement

Pichia pastors is a popular yeast strain for heterologous protein expression. For example P. pastors has been used to express over 500 proteins. Heterologous protein expression allows a host organism to express a protein that is not normally in a host organism. P. pastors is an attractive host organism to use because it doesn't secrete a lot of its own protein, which means that little purification of the target protein needs to be done, once your target protein is secreted. Though it is advantageous to use P. pastors, there are some drawbacks. Some proteins do not secrete very well from P. pastors, meaning the protein stays trapped in the cell of the yeast. The goal of this project is to isolate mutants of P. pastors which are super secretors, which are those that secrete more efficiently than the wild type. In order to accomplish this task, a reporter protein has to be set up to aid in screening the mutants. Prochymosin is used as the reporter protein because it has been used to isolate super secretors of Saccharomyces cerevisiae, and should be easy to use. In this project, prochymosin is expressed from the pGAP promoter. A pGAPza-prochymosin reporter plasmid was constructed and electroporated into competent yGS115 cells, which is a strain of P. pastors. The cells were then grown on Zeocin plates to select for colonies that had picked up the plasmid. These colonies were streaked for single colonie and assayed by Western to confirm that the P. pastors clones expressed and secreted the prochymosin. Once we understand how well the reporter protein works in the wild type, data can be gathered from the mutant strains and compared to see if it is a super secretor.

Location

Pacific Geosciences Center

Start Date

26-4-2003 9:00 AM

End Date

26-4-2003 5:00 PM

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Apr 26th, 9:00 AM Apr 26th, 5:00 PM

Expression and secretion of prochymosin in Pichia pastoris.

Pacific Geosciences Center

Pichia pastors is a popular yeast strain for heterologous protein expression. For example P. pastors has been used to express over 500 proteins. Heterologous protein expression allows a host organism to express a protein that is not normally in a host organism. P. pastors is an attractive host organism to use because it doesn't secrete a lot of its own protein, which means that little purification of the target protein needs to be done, once your target protein is secreted. Though it is advantageous to use P. pastors, there are some drawbacks. Some proteins do not secrete very well from P. pastors, meaning the protein stays trapped in the cell of the yeast. The goal of this project is to isolate mutants of P. pastors which are super secretors, which are those that secrete more efficiently than the wild type. In order to accomplish this task, a reporter protein has to be set up to aid in screening the mutants. Prochymosin is used as the reporter protein because it has been used to isolate super secretors of Saccharomyces cerevisiae, and should be easy to use. In this project, prochymosin is expressed from the pGAP promoter. A pGAPza-prochymosin reporter plasmid was constructed and electroporated into competent yGS115 cells, which is a strain of P. pastors. The cells were then grown on Zeocin plates to select for colonies that had picked up the plasmid. These colonies were streaked for single colonie and assayed by Western to confirm that the P. pastors clones expressed and secreted the prochymosin. Once we understand how well the reporter protein works in the wild type, data can be gathered from the mutant strains and compared to see if it is a super secretor.