Directed evolution for improved secretion of cancer-testis antigen NY-ESO-1 from yeast.

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.

• Authors: Andrea Piatesi, Shanshan W Howland, James A Rakestraw, Christoph Renner, Neil Robson, Jonathan Cebon, Eugene Maraskovsky, Gerd Ritter, Lloyd Old, K Dane Wittrup
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 48 Issue 2 Pg. 232-42 (Aug 2006) ISSN: 1046-5928 [Print] United States
• PMID: 16563796 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antigens, Neoplasm
• CTAG1B protein, human
• Membrane Proteins
• Recombinant Proteins

Topics

• Amino Acid Sequence
• Antigens, Neoplasm (genetics, isolation & purification)
• Directed Molecular Evolution (methods)
• Escherichia coli
• Genetic Techniques
• Humans
• Male
• Membrane Proteins (genetics, isolation & purification, metabolism)
• Models, Molecular
• Molecular Sequence Data
• Recombinant Proteins (biosynthesis, genetics, isolation & purification, metabolism)
• Saccharomyces cerevisiae (genetics, metabolism)
• Sequence Homology, Amino Acid