Directed evolution for improved secretion of cancer-testis antigen NY-ESO-1 from yeast.
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.
Division of Biological Engineering, Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, 02139, USA.
Andrea
A
Piatesi
Shanshan W
SW
Howland
James A
JA
Rakestraw
Christoph
C
Renner
Neil
N
Robson
Jonathan
J
Cebon
Eugene
E
Maraskovsky
Gerd
G
Ritter
Lloyd
L
Old
K Dane
KD
Wittrup
D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000235:genetics* / Q000302:isolation & purification; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli; D005821:Genetic Techniques; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000951:Antigens, Neoplasm; C104948:CTAG1B protein, human; D008565:Membrane Proteins; D011994:Recombinant Proteins