전체메뉴
검색
Article Search

JMB Journal of Microbiolog and Biotechnology

QR Code QR Code

Research article

Clinical Microbiology and Biomedical Sciences

Optimization of Yeast Surface-Displayed cDNA Library Screening for Low Abundance Targets

Juhyung Kim 1, Hyung Kyu Kim 1, Hye Jeong Jang 1, Eunkyung Kim 1 and Moon Kyu Kim 1*

Department of Immunology, School of Medicine, Kyungpook National University, Daegu 700-422, Republic of Korea

Received: September 25, 2014; Accepted: November 10, 2014

J. Microbiol. Biotechnol. 2015; 25(4): 547-553

Published April 28, 2015 https://doi.org/10.4014/jmb.1409.09079

Copyright © The Korean Society for Microbiology and Biotechnology.

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.

Keywords

cDNA library, yeast surface display, tumor-associated antigen, hepatocellular carcinoma

References

  1. Bidlingmaier S, He J, Wang Y, An F, Feng J, Barbone D, et al. 2009. 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.
    Pubmed PMC CrossRef
  2. Bidlingmaier S, Liu B. 2006. Construction and application of a yeast surface-displayed human cDNA library to identify post-translational modification-dependent protein-protein interactions. Mol. Cell. Proteomics 5: 533-540.
    Pubmed CrossRef
  3. Bidlingmaier S, Liu B. 2011. Construction of yeast surfacedisplayed cDNA libraries. Methods Mol. Biol. 729: 199-210.
    Pubmed PMC CrossRef
  4. Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD. 2006. Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1: 755-768.
    Pubmed CrossRef
  5. Daugherty PS, Iverson BL, Georgiou G. 2000. Flow cytometric screening of cell-based libraries. J. Immunol. Methods 243:211-227.
    CrossRef
  6. Davis C, Barvish Z, Gitelman I. 2007. A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA. BMC Genomics 8: 363.
    Pubmed PMC CrossRef
  7. Foung S, Perkins S, Kafadar K, Gessner P, Zimmermann U. 1990. Development of microfusion techniques to generate human hybridomas. J. Immunol. Methods 134: 35-42.
    CrossRef
  8. Foung SK, Perkins S. 1989. Electric field-induced cell fusion and human monoclonal antibodies. J. Immunol. Methods 116:117-122.
    CrossRef
  9. Galluzzi L, Vacchelli E, Fridman WH, Galon J, SautsFridman C, Tartour E, et al. 2012. Trial watch: monoclonal antibodies in cancer therapy. Oncoimmunology 1: 28.
    Pubmed PMC CrossRef
  10. Howland SW, Poh C-M, Rnia L. 2011. Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioatemodified primers. Anal. Biochem. 416: 141-143.
    Pubmed CrossRef
  11. Jack HM, Beck-Engeser G, Lee G, Wofsy D, Wabl M. 1992. Tumorigenesis mediated by an antigen receptor. Proc. Natl. Acad. Sci. USA 89: 8482-8486.
    Pubmed PMC CrossRef
  12. Jung V, Fischer E, Imig J, Kleber S, Nuber N, Reinshagen F, et al. 2010. Yeast-based identification of prostate tumor antigens provides an effective vaccine platform. Anticancer Res. 30: 895-902.
    Pubmed
  13. Kieke MC, Cho BK, Boder ET, Kranz DM, Wittrup KD. 1997. Isolation of anti-T cell receptor scFv mutants by yeast surface display. Protein Eng. 10: 1303-1310.
    Pubmed CrossRef
  14. Kim M, Choi HY, Choi YS, Kim J, Kim Y. 2007. Optimized serological isolation of lung-cancer-associated antigens from a yeast surface-expressed cDNA library. J. Microbiol. Biotechnol. 17: 993.
    Pubmed
  15. Kondo A, Ueda M. 2004. Yeast cell-surface display −applications of molecular display. Appl. Microbiol. Biotechnol. 64: 28-40.
    Pubmed CrossRef
  16. Pepper LR, Cho YK, Boder ET, Shusta EV. 2008. A decade of yeast surface display technology: where are we now? Comb. Chem. High Throughput Screen. 11: 127.
    Pubmed PMC CrossRef
  17. Van Antwerp JJ, Wittrup KD. 2000. Fine affinity discrimination by yeast surface display and flow cytometry. Biotechnol. Prog. 16: 31-37.
    Pubmed CrossRef
  18. Wadle A, Mischo A, Imig J, Wllner B, Hensel D, Wtzig K, et al. 2005. Serological identification of breast cancer-related antigens from a Saccharomyces cerevisiae surface display library. Int. J. Cancer 117: 104-113.
    Pubmed CrossRef
  19. Yeung YA, Wittrup KD. 2002. Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture. Biotechnol. Prog. 18: 212-220.
    Pubmed CrossRef
  20. Yu X, McGraw PA, House FS, Crowe JE Jr. 2008. An optimized electrofusion-based protocol for generating virusspecific human monoclonal antibodies. J. Immunol. Methods 336: 142-151.
    Pubmed PMC CrossRef

Related articles in JMB

More Related Articles

Article

Research article

J. Microbiol. Biotechnol. 2015; 25(4): 547-553

Published online April 28, 2015 https://doi.org/10.4014/jmb.1409.09079

Copyright © The Korean Society for Microbiology and Biotechnology.

Optimization of Yeast Surface-Displayed cDNA Library Screening for Low Abundance Targets

Juhyung Kim 1, Hyung Kyu Kim 1, Hye Jeong Jang 1, Eunkyung Kim 1 and Moon Kyu Kim 1*

Department of Immunology, School of Medicine, Kyungpook National University, Daegu 700-422, Republic of Korea

Received: September 25, 2014; Accepted: November 10, 2014

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.

Keywords: cDNA library, yeast surface display, tumor-associated antigen, hepatocellular carcinoma

References

  1. Bidlingmaier S, He J, Wang Y, An F, Feng J, Barbone D, et al. 2009. 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.
    Pubmed KoreaMed CrossRef
  2. Bidlingmaier S, Liu B. 2006. Construction and application of a yeast surface-displayed human cDNA library to identify post-translational modification-dependent protein-protein interactions. Mol. Cell. Proteomics 5: 533-540.
    Pubmed CrossRef
  3. Bidlingmaier S, Liu B. 2011. Construction of yeast surfacedisplayed cDNA libraries. Methods Mol. Biol. 729: 199-210.
    Pubmed KoreaMed CrossRef
  4. Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD. 2006. Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1: 755-768.
    Pubmed CrossRef
  5. Daugherty PS, Iverson BL, Georgiou G. 2000. Flow cytometric screening of cell-based libraries. J. Immunol. Methods 243:211-227.
    CrossRef
  6. Davis C, Barvish Z, Gitelman I. 2007. A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA. BMC Genomics 8: 363.
    Pubmed KoreaMed CrossRef
  7. Foung S, Perkins S, Kafadar K, Gessner P, Zimmermann U. 1990. Development of microfusion techniques to generate human hybridomas. J. Immunol. Methods 134: 35-42.
    CrossRef
  8. Foung SK, Perkins S. 1989. Electric field-induced cell fusion and human monoclonal antibodies. J. Immunol. Methods 116:117-122.
    CrossRef
  9. Galluzzi L, Vacchelli E, Fridman WH, Galon J, SautsFridman C, Tartour E, et al. 2012. Trial watch: monoclonal antibodies in cancer therapy. Oncoimmunology 1: 28.
    Pubmed KoreaMed CrossRef
  10. Howland SW, Poh C-M, Rnia L. 2011. Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioatemodified primers. Anal. Biochem. 416: 141-143.
    Pubmed CrossRef
  11. Jack HM, Beck-Engeser G, Lee G, Wofsy D, Wabl M. 1992. Tumorigenesis mediated by an antigen receptor. Proc. Natl. Acad. Sci. USA 89: 8482-8486.
    Pubmed KoreaMed CrossRef
  12. Jung V, Fischer E, Imig J, Kleber S, Nuber N, Reinshagen F, et al. 2010. Yeast-based identification of prostate tumor antigens provides an effective vaccine platform. Anticancer Res. 30: 895-902.
    Pubmed
  13. Kieke MC, Cho BK, Boder ET, Kranz DM, Wittrup KD. 1997. Isolation of anti-T cell receptor scFv mutants by yeast surface display. Protein Eng. 10: 1303-1310.
    Pubmed CrossRef
  14. Kim M, Choi HY, Choi YS, Kim J, Kim Y. 2007. Optimized serological isolation of lung-cancer-associated antigens from a yeast surface-expressed cDNA library. J. Microbiol. Biotechnol. 17: 993.
    Pubmed
  15. Kondo A, Ueda M. 2004. Yeast cell-surface display −applications of molecular display. Appl. Microbiol. Biotechnol. 64: 28-40.
    Pubmed CrossRef
  16. Pepper LR, Cho YK, Boder ET, Shusta EV. 2008. A decade of yeast surface display technology: where are we now? Comb. Chem. High Throughput Screen. 11: 127.
    Pubmed KoreaMed CrossRef
  17. Van Antwerp JJ, Wittrup KD. 2000. Fine affinity discrimination by yeast surface display and flow cytometry. Biotechnol. Prog. 16: 31-37.
    Pubmed CrossRef
  18. Wadle A, Mischo A, Imig J, Wllner B, Hensel D, Wtzig K, et al. 2005. Serological identification of breast cancer-related antigens from a Saccharomyces cerevisiae surface display library. Int. J. Cancer 117: 104-113.
    Pubmed CrossRef
  19. Yeung YA, Wittrup KD. 2002. Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture. Biotechnol. Prog. 18: 212-220.
    Pubmed CrossRef
  20. Yu X, McGraw PA, House FS, Crowe JE Jr. 2008. An optimized electrofusion-based protocol for generating virusspecific human monoclonal antibodies. J. Immunol. Methods 336: 142-151.
    Pubmed KoreaMed CrossRef