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Updated: Aug 9, 2026
Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
Directed evolution for improved secretion of cancer-testis antigen NY-ESO-1 from yeast
Andrea Piatesi1, Shanshan W Howland, James A Rakestraw
1Division of Biological Engineering, Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, 02139, USA.
Abstract:
NY-ESO-1 is a highly immunogenic tumor antigen and a promising vaccine candidate in cancer immunotherapy. Access to purified protein both for vaccine formulations and for monitoring antigen-specific immune responses is vital to vaccine development. Currently available recombinant Escherichia coli-derived NY-ESO-1 is isolated from inclusion bodies as a complex protein mixture and efforts to improve the purity of this antigen are required, especially for later-stage clinical trials. Using yeast cell surface display and fluorescence activated cell sorting techniques, we have engineered an NY-ESO-1 variant (NY-ESO-L5; C(75)A C(76)A C(78)A L(153)H) with a 100x improved display level on yeast compared to the wild-type protein. This mutant can be effectively produced as an Aga2p-fusion and purified in soluble form directly from the yeast cell wall. In the process, we have identified the epitope recognized by anti-NY-ESO-1 mAb E978 (79-87, GARGPESRL). The availability of an alternative expression host for this important antigen will help avoid artifactual false positive tests of patient immune response due to reaction against expression-host-specific contaminants.
Insights
Researchers engineered a novel NY-ESO-1 variant for improved cancer immunotherapy vaccine development. This enhanced protein, produced in yeast, offers higher purity and aids in accurate immune response monitoring.
Area of Science:
- Immunology
- Biotechnology
- Cancer Research
Background:
- NY-ESO-1 is a key tumor antigen for cancer immunotherapy vaccines.
- Current recombinant NY-ESO-1 from E. coli has purity issues, hindering clinical trials.
- Improved antigen purification is crucial for vaccine formulation and immune response monitoring.
Purpose of the Study:
- To engineer a highly displayable and purifiable NY-ESO-1 variant using yeast cell surface display.
- To identify the specific epitope recognized by the anti-NY-ESO-1 mAb E978.
- To provide an alternative expression system for NY-ESO-1 to improve vaccine development and diagnostic accuracy.
Main Methods:
- Yeast cell surface display and fluorescence-activated cell sorting (FACS) were employed.
- Mutagenesis was used to create an NY-ESO-1 variant (NY-ESO-L5).
- The variant was produced as an Aga2p-fusion and purified from the yeast cell wall.
Main Results:
- An NY-ESO-1 variant (NY-ESO-L5) with 100x improved yeast display was engineered.
- This variant was successfully produced and purified in soluble form.
- The epitope for anti-NY-ESO-1 mAb E978 was identified as residues 79-87 (GARGPESRL).
Conclusions:
- The engineered yeast-expressed NY-ESO-1 variant offers a superior alternative for vaccine development.
- This method enhances antigen purity, crucial for reliable cancer immunotherapy.
- Accurate epitope identification aids in precise immune response assessment and avoids host-specific contaminants.
