Optimization of Yeast Surface-Displayed cDNA Library Screening for Low Abundance Targets
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The yeast surface-displayed cDNA library has been used to identify unknown antigens. However, when unknown target antigens show moderate-to-low abundance, some modifications are needed in the screening process. In this study, a directional random-primed cDNA library was used to increase the number of candidates for the unknown antigen. To avoid the loss of target yeast clones that express proteins at a low frequency in the cDNA library, a comprehensive monitoring system based on magnetic-activated cell sorting, fluorescence-activated cell sorting, and immunofluorescence was established, and a small number of target yeast cells was successfully enriched. These results showed that our optimized method has potential application for identifying rare unknown antigens of the human monoclonal antibody.
Abstract
The yeast surface-displayed cDNA library has been used to identify unknown antigens. However, when unknown target antigens show moderate-to-low abundance, some modifications are needed in the screening process. In this study, a directional random-primed cDNA library was used to increase the number of candidates for the unknown antigen. To avoid the loss of target yeast clones that express proteins at a low frequency in the cDNA library, a comprehensive monitoring system based on magnetic-activated cell sorting, fluorescence-activated cell sorting, and immunofluorescence was established, and a small number of target yeast cells was successfully enriched. These results showed that our optimized method has potential application for identifying rare unknown antigens of the human monoclonal antibody.
참고문헌 (20)
- Bidlingmaier S, "Identification of MCAM", CD146 as the target antigen of a human monoclonal antibody that recognizes both epithelioid and sarcomatoid types of mesothelioma. Cancer Res. 69 : 1570-1577, 2009, pp.69, 1570~1577
- Bidlingmaier S, "Construction and application of a yeast surface-displayed human cDNA library to identify post-translational modification-dependent protein-protein interactions", Mol. Cell. Proteomics, 5, pp.533~540 (2006)
- Bidlingmaier S, "Construction of yeast surfacedisplayed cDNA libraries", Methods Mol. Biol, 729, pp.199~210 (2011)
- Chao G, "Isolating and engineering human antibodies using yeast surface display", Nat. Protoc, 1, pp.755~768 (2006)
- Daugherty PS, "Flow cytometric screening of cell-based libraries", J. Immunol. Methods, 243, pp.211~227 (2000)
- Davis C, "A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA", BMC Genomics, 8, pp.363~ (2007)
- Foung S, "Development of microfusion techniques to generate human hybridomas", J. Immunol. Methods, 134, pp.35~42 (1990)
- Foung SK, "Electric field-induced cell fusion and human monoclonal antibodies", J. Immunol. Methods, 116, pp.117~122 (1989)
- Galluzzi L, "Trial watch: monoclonal antibodies in cancer therapy", Oncoimmunology, 1, pp.28~ (2012)
- Howland SW, "Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioatemodified primers", Anal. Biochem, 416, pp.141~143 (2011)
- Jack HM, "Tumorigenesis mediated by an antigen receptor", Proc. Natl. Acad. Sci. USA, 89, pp.8482~8486 (1992)
- Jung V, "Yeast-based identification of prostate tumor antigens provides an effective vaccine platform", Anticancer Res, 30, pp.895~902 (2010)
- Kieke MC, "Isolation of anti-T cell receptor scFv mutants by yeast surface display", Protein Eng, 10, pp.1303~1310 (1997)
- KIM, MIN-SOO, "Optimized Serological Isolation of Lung-Cancer-associated Antigens from aYeast Surface-expressed cDNA Library", Journal of Microbiology and Biotechnology, 17(6), pp.993~1001 (2007), 한국미생물·생명공학회
- Kondo A, "Yeast cell-surface display −applications of molecular display", Appl. Microbiol. Biotechnol, 64, pp.28~40 (2004)
- Pepper LR, "A decade of yeast surface display technology: where are we now?", Comb. Chem. High Throughput Screen, 11, pp.127~ (2008)
- Van Antwerp JJ, "Fine affinity discrimination by yeast surface display and flow cytometry", Biotechnol. Prog, 16, pp.31~37 (2000)
- Wadle A, "Serological identification of breast cancer-related antigens from a Saccharomyces cerevisiae surface display library", Int. J. Cancer, 117, pp.104~113 (2005)
- Yeung YA, "Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture", Biotechnol. Prog, 18, pp.212~220 (2002)
- Yu X, "An optimized electrofusion-based protocol for generating virusspecific human monoclonal antibodies", J. Immunol. Methods, 336, pp.142~151 (2008)