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An optimized yeast display strategy for efficient scFv antibody selection using ribosomal skipping system and thermo resistant yeast

  • Yanrong Jia
  • Ping Ren
  • Shixin Duan
  • Pei Zeng
  • Debao Xie
  • Fanli ZengEmail author
Original Research Paper
  • 34 Downloads

Abstract

Objectives

Establish a method to restrict unexpected fragments including stop codons in scFv library and generate a thermo resistant strain for screening of thermal stable scFv sequences.

Results

Here, we have constructed a T2A–Leu2 system for selection of yeast surface display libraries that blocks amplification of “stop codon” plasmids within the library, thereby increasing the quality of the library and efficiency of the selection screen. Also, we generated a temperature-resistant yeast strain, TR1, and validated its combined use with T2A–Leu2 for efficient screening. Thus, we developed a general approach for a fast and efficient screening of scFv libraries using a ribosomal skipping system and thermo-resistant yeast.

Conclusions

The method highlights the utility of the T2A–Leu2-based ribosomal skipping strategy for increasing the quality of the input library for selection, along with an optimized selection protocol based on thermo-resistant yeast cells.

Keywords

Yeast surface display scFv antibody Ribosomal skipping T2A Antibody screen 

Notes

Supporting information

Supplementary Table 1—Nter-SfiI and scFv DNA sequences used in this study.

Supplementary Table 2—Oligos used in this study.

Supplementary Figure 1—Map of pCTcon2-derived plasmid pDJ21.

Supplementary Figure 2—Map of T2A-Leu2 plasmid pDJ22.

Funding

This work was supported by a Starting Grant from Hebei Agricultural University (to Fanli Zeng; Grant No. 2018KYYJ01) and by a grant from the National Natural Science Foundation of China (to Fanli Zeng; Grant No. 31801039).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

10529_2019_2710_MOESM1_ESM.docx (17 kb)
Supplementary material 1 (DOCX 16 kb)
10529_2019_2710_MOESM2_ESM.tiff (413 kb)
Supplementary material 2 (TIFF 412 kb)
10529_2019_2710_MOESM3_ESM.tiff (342 kb)
Supplementary material 3 (TIFF 342 kb)

References

  1. Angelini A, Chen TF, de Picciotto S, Yang NJ, Tzeng A, Santos MS, Van Deventer JA, Traxlmayr MW, Wittrup KD (2015) Protein engineering and selection using yeast surface display. Methods Mol Biol 1319:3–36CrossRefGoogle Scholar
  2. Caucheteur D, Robin G, Parez V, Martineau P (2018) Construction of a synthetic antibody gene library for the selection of intrabodies and antibodies. Methods Mol Biol 1701:239–253CrossRefGoogle Scholar
  3. Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD (2006) Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1:755–768CrossRefGoogle Scholar
  4. Cherf GM, Cochran JR (2015) Applications of yeast surface display for protein engineering. Methods Mol Biol 1319:155–175CrossRefGoogle Scholar
  5. Chng J, Wang T, Nian R, Lau A, Hoi KM, Ho SC, Gagnon P, Bi X, Yang Y (2015) Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells. Mabs-Austin 7:403–412CrossRefGoogle Scholar
  6. Cruz-Teran CA, Tiruthani K, Mischler A, Rao BM (2017) Inefficient ribosomal skipping enables simultaneous secretion and display of proteins in Saccharomyces cerevisiae. ACS Synth Biol 6:2096–2107CrossRefGoogle Scholar
  7. de Felipe P, Hughes LE, Ryan MD, Brown JD (2003) Co-translational, intraribosomal cleavage of polypeptides by the foot-and-mouth disease virus 2A peptide. J Biol Chem 278:11441–11448CrossRefGoogle Scholar
  8. Dusseaux MMF (2018) Cells for immunotherapy engineered for targeting cd38 antigen and for cd38 gene inactivation. United States Patent, 20180236053Google Scholar
  9. Eeckhout D, Fiers E, Sienaert R, Snoeck V, Depicker A, De Jaeger G (2000) Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana. Eur J Biochem 267:6775–6783CrossRefGoogle Scholar
  10. Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JM, Yeung YA, Cochran JR, Heinzelman P, Colby D, Swers J, Graff C, Wiley HS, Wittrup KD (2003) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol 21:163–170CrossRefGoogle Scholar
  11. Fernandes JC (2018) Therapeutic application of antibody fragments in autoimmune diseases: current state and prospects. Drug Discov Today 23:1996–2002CrossRefGoogle Scholar
  12. Grzeschik J, Hinz SC, Konning D, Pirzer T, Becker S, Zielonka S, Kolmar H (2017) A simplified procedure for antibody engineering by yeast surface display: coupling display levels and target binding by ribosomal skipping. Biotechnol J 12:1600454CrossRefGoogle Scholar
  13. Hu Y, Jia Y, Zhao X, Yang Z, Hao Z, Dong J, Zeng F (2019) A new strategy for seamless gene editing and marker recycling in Saccharomyces cerevisiae using lethal effect of Cwp1. Microbiologyopen 8:e750Google Scholar
  14. Hua CK, Gacerez AT, Sentman CL, Ackerman ME (2017) Development of unique cytotoxic chimeric antigen receptors based on human scFv targeting B7H6. Protein Eng Des Sel 30:713–721CrossRefGoogle Scholar
  15. Jeong MY, Rutter J, Chou DH (2019) Display of single-chain insulin-like peptides on a yeast surface. Biochemistry 58:182–188CrossRefGoogle Scholar
  16. Romao E, Poignavent V, Vincke C, Ritzenthaler C, Muyldermans S, Monsion B (2018) Construction of high-quality camel immune antibody libraries. Methods Mol Biol 1701:169–187CrossRefGoogle Scholar
  17. Rosowski S, Becker S, Toleikis L, Valldorf B, Grzeschik J, Demir D, Willenbucher I, Gaa R, Kolmar H, Zielonka S, Krah S (2018) A novel one-step approach for the construction of yeast surface display Fab antibody libraries. Microb Cell Fact 17:3CrossRefGoogle Scholar
  18. Scholler N (2012) Selection of antibody fragments by yeast display. Methods Mol Biol 907:259–280CrossRefGoogle Scholar
  19. Shave S, Mann S, Koszela J, Kerr A, Auer M (2018) PuLSE: quality control and quantification of peptide sequences explored by phage display libraries. PLoS ONE 13:e193332CrossRefGoogle Scholar
  20. Sun Y, Ban B, Bradbury A, Ansari GA, Blake DA (2016) Combining yeast display and competitive FACS to select rare hapten-specific clones from recombinant antibody libraries. Anal Chem 88:9181–9189CrossRefGoogle Scholar
  21. Van Deventer JA, Wittrup KD (2014) Yeast surface display for antibody isolation: library construction, library screening, and affinity maturation. Methods Mol Biol 1131:151–181CrossRefGoogle Scholar
  22. Yuan X, Chen X, Yang M, Hu J, Yang W, Chen T, Wang Q, Zhang X, Lin R, Zhao A (2016) Efficient construct of a large and functional scFv yeast display library derived from the ascites B cells of ovarian cancer patients by three-fragment transformation-associated recombination. Appl Microbiol Biotechnol 100:4051–4061CrossRefGoogle Scholar
  23. Zeng F, Hua Y, Liu X, Liu S, Lao K, Zhang Z, Kong D (2018) Gpn2 and rba50 directly participate in the assembly of the rpb3 subcomplex in the biogenesis of RNA polymerase II. Mol Cell Biol 38:18–91Google Scholar
  24. Zhang Z, Ren P, Vashisht AA, Wohlschlegel JA, Quintana DG, Zeng F (2017) Cdk1-interacting protein Cip1 is regulated by the S phase checkpoint in response to genotoxic stress. Genes Cells 22:850–860CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.College of Life SciencesHebei Agricultural UniversityBaodingChina
  2. 2.Shanghai Institute for Advanced Immunochemical StudiesShanghaiTech UniversityShanghaiChina

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