Skip to content
2000
  1. Home
  2. A-Z Publications
  3. Combinatorial Chemistry & High Throughput Screening
  4. Fast Track Articles
  5. Fast Track Article
image of Rapid Screening and Effective Rabbit-Derived Fab Antibodies Production Based on Yeast Surface Display

Abstract

Introduction/Objective

Antibodies have broad applications in various fields, such as biology and medicine. The screening and preparation of highly specific and sensitive antibodies are essential research areas. Several techniques for the preparation of mouse-derived antibodies have been developed, but limited studies on rabbit-derived antibodies with a broader antibody profile and easier humanization are reported. An improved yeast surface display technique was used for rapid screening of rabbit-derived Fab antibodies.

Methods

After RNA extraction from peripheral rabbit blood, a cDNA library was obtained by reverse transcription. After recombinant vector construction, the expressed sequence in the form of Fab antibody structure was fused to the N-terminal end of Aga2p in the vector; a bidirectional promoter was inserted and successfully expressed in brewer's yeast EBY100. In addition, sequences, such as leucine zipper and inulinase signal peptide (INU), were inserted into the recombinant vector to improve the expression and stability of Fab antibody further.

Results

A biotin-labeled salbutamol marker was synthesized, and two rabbit-derived salbutamol-Fab antibodies were screened in three weeks using fluorescence-activated cell sorting (FACS).

Conclusion

After antigen-binding kinetic studies, the screened antibodies demonstrated good affinity and specificity.

© 2025 Bentham Science Publishers
Loading

Article metrics loading...

/content/journals/cchts/10.2174/0113862073352395250211052148
2025-02-24
2025-06-18

  • From This Site

    /content/journals/cchts/10.2174/0113862073352395250211052148
    dcterms_title,dcterms_subject,pub_keyword
    -contentType:Contributor -contentType:Concept -contentType:Institution
    10
    5
Loading full text...

Full text loading...

References

  1. Sujan K. Dhar M.D. Engineering antibodies. J. Indian Inst. Sci. 2018 98 1
    [Google Scholar]
  2. Manabe S. Yamaguchi Y. Antibody glycoengineering and homogeneous antibody‐drug conjugate preparation. Chem. Rec. 2021 21 11 3005 3014 10.1002/tcr.202100054 33886147
    [Google Scholar]
  3. Madan B. Reddem E.R. Wang P. Casner R.G. Nair M.S. Huang Y. Fahad A.S. de Souza M.O. Banach B.B. López Acevedo S.N. Pan X. Nimrania R. Teng I.T. Bahna F. Zhou T. Zhang B. Yin M.T. Ho D.D. Kwong P.D. Shapiro L. DeKosky B.J. Antibody screening at reduced pH enables preferential selection of potently neutralizing antibodies targeting SARS‐CoV ‐2. AIChE J. 2021 67 12 e17440 10.1002/aic.17440 34898670
    [Google Scholar]
  4. Parola C. Neumeier D. Reddy S.T. Integrating high‐throughput screening and sequencing for monoclonal antibody discovery and engineering. Immunology 2018 153 1 31 41 10.1111/imm.12838 28898398
    [Google Scholar]
  5. Ilinykh P.A. Graber J. Kuzmina N.A. Huang K. Ksiazek T.G. Crowe J.E. Jr Bukreyev A. Ebolavirus chimerization for the development of a mouse model for screening of bundibugyo-specific antibodies. J. Infect. Dis. 2018 218 Suppl. 5 S418 S422 30060231
    [Google Scholar]
  6. Kato M. Hanyu Y. Single-step colony assay for screening antibody libraries. J. Biotechnol. 2017 255 1 8 10.1016/j.jbiotec.2017.06.010 28641985
    [Google Scholar]
  7. Bie Z. Xing R. He X. Ma Y. Chen Y. Liu Z. Precision imprinting of glycopeptides for facile preparation of glycan-specific artificial antibodies. Anal. Chem. 2018 90 16 9845 9852 10.1021/acs.analchem.8b01903 30036038
    [Google Scholar]
  8. Yaneva M.Y. Ivanov Y.L. Godjevargova T.I. Preparation of polyclonal antibodies with application for an organophosphorus pesticide immunoassay. Anal. Lett. 2017 50 8 1307 1324 10.1080/00032719.2016.1221417
    [Google Scholar]
  9. Laustsen A.H. Greiff V. Karatt-Vellatt A. Muyldermans S. Jenkins T.P. Animal immunization, in vitro display technologies, and machine learning for antibody discovery. Trends Biotechnol. 2021 39 12 1263 1273 10.1016/j.tibtech.2021.03.003 33775449
    [Google Scholar]
  10. Prabakaran P. Rao S.P. Wendt M. Animal immunization merges with innovative technologies: A new paradigm shift in antibody discovery. MAbs 2021 13 1 1924347 10.1080/19420862.2021.1924347 33947305
    [Google Scholar]
  11. Greenfield E.A. Standard immunization of rabbits. Cold Spring Harb. Protoc. 2020 2020 9 pdb.prot100305 10.1101/pdb.prot100305 32873729
    [Google Scholar]
  12. Rossi S. Laurino L. Furlanetto A. Chinellato S. Orvieto E. Canal F. Facchetti F. Dei Tos A.P. Rabbit monoclonal antibodies: A comparative study between a novel category of immunoreagents and the corresponding mouse monoclonal antibodies. Am. J. Clin. Pathol. 2005 124 2 295 302 10.1309/NR8HN08GDPVEMU08 16040303
    [Google Scholar]
  13. Spieker-Polet H. Sethupathi P. Yam P.C. Knight K.L. Rabbit monoclonal antibodies: Generating a fusion partner to produce rabbit-rabbit hybridomas. Proc. Natl. Acad. Sci. USA 1995 92 20 9348 9352 10.1073/pnas.92.20.9348 7568130
    [Google Scholar]
  14. Gary C. Making and Using Antibodies: A Practical Handbook 2nd ed CRC Press 2013 7 29
    [Google Scholar]
  15. Weber J. Peng H. Rader C. From rabbit antibody repertoires to rabbit monoclonal antibodies. Exp. Mol. Med. 2017 49 3 e305 10.1038/emm.2017.23 28336958
    [Google Scholar]
  16. Kennedy P.J. Oliveira C. Granja P.L. Sarmento B. Monoclonal antibodies: Technologies for early discovery and engineering. Crit. Rev. Biotechnol. 2018 38 3 394 408 10.1080/07388551.2017.1357002 28789584
    [Google Scholar]
  17. Phua S.X. Chan K.F. Su C.T.T. Poh J.J. Gan S.K.E. Perspective: The promises of a holistic view of proteins—impact on antibody engineering and drug discovery. Biosci. Rep. 2019 39 1 BSR20181958 10.1042/BSR20181958 30630879
    [Google Scholar]
  18. Sargunas P.R. Spangler J.B. Full speed AHEAD in antibody discovery. Nat. Chem. Biol. 2021 17 10 1011 1012 10.1038/s41589‑021‑00838‑y 34211163
    [Google Scholar]
  19. Gaa R. Menang-Ndi E. Pratapa S. Nguyen C. Kumar S. Doerner A. Versatile and rapid microfluidics-assisted antibody discovery. MAbs 2021 13 1 1978130 10.1080/19420862.2021.1978130 34586015
    [Google Scholar]
  20. Boder E.T. Wittrup K.D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 1997 15 6 553 557 10.1038/nbt0697‑553 9181578
    [Google Scholar]
  21. Salema V. Fernández L.Á. Escherichia coli surface display for the selection of nanobodies. Microb. Biotechnol. 2017 10 6 1468 1484 10.1111/1751‑7915.12819 28772027
    [Google Scholar]
  22. Elison G.L. Xue Y. Song R. Acar M. Insights into bidirectional gene expression control using the canonical GAL1/GAL10 promoter. Cell Rep. 2018 25 3 737 748.e4 10.1016/j.celrep.2018.09.050 30332652
    [Google Scholar]
  23. Partow S. Siewers V. Bjørn S. Nielsen J. Maury J. Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast 2010 27 11 955 964 10.1002/yea.1806 20625983
    [Google Scholar]
  24. Urit T. Löser C. Wunderlich M. Bley T. Formation of ethyl acetate by Kluyveromyces marxianus on whey: Studies of the ester stripping. Bioprocess Biosyst. Eng. 2011 34 5 547 559 10.1007/s00449‑010‑0504‑9 21191616
    [Google Scholar]
  25. Pal Y. Khushoo A. Mukherjee K.J. Process optimization of constitutive human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expression in Pichia pastoris fed-batch culture. Appl. Microbiol. Biotechnol. 2006 69 6 650 657 10.1007/s00253‑005‑0018‑6 15983807
    [Google Scholar]
  26. Chung B.H. Nam S.W. Kim B.M. Park Y.H. Highly efficient secretion of heterologous proteins from Saccharomyces cerevisiae using inulinase signal peptides. Biotechnol. Bioeng. 1996 49 4 473 479 10.1002/(SICI)1097‑0290(19960220)49:4<473::AID‑BIT15>3.0.CO;2‑B 18623603
    [Google Scholar]
  27. Sivelle C. Sierocki R. Ferreira-Pinto K. Simon S. Maillere B. Nozach H. Fab is the most efficient format to express functional antibodies by yeast surface display. MAbs 2018 10 5 720 729 10.1080/19420862.2018.1468952 29708852
    [Google Scholar]
  28. Gupta S.K. Shukla P. Microbial platform technology for recombinant antibody fragment production: A review. Crit. Rev. Microbiol. 2017 43 1 31 42 10.3109/1040841X.2016.1150959 27387055
    [Google Scholar]
  29. Alber T. Structure of the leucine zipper. Curr. Opin. Genet. Dev. 1992 2 2 205 210 10.1016/S0959‑437X(05)80275‑8 1638114
    [Google Scholar]
  30. Barnes B.E. Jenkins T.A. Stein L.M. Mathers R.T. Wicaksana M. Pasquinelli M.A. Savin D.A. Synthesis and characterization of a leucine-based block co-polypeptide: The effect of the leucine zipper on self-assembly. Biomacromolecules 2020 21 6 2463 2472 10.1021/acs.biomac.0c00420 32378896
    [Google Scholar]
  31. Bogen J.P. Storka J. Yanakieva D. Fiebig D. Grzeschik J. Hock B. Kolmar H. Isolation of common light chain antibodies from immunized chickens using yeast biopanning and fluorescence‐activated cell sorting. Biotechnol. J. 2021 16 3 2000240 10.1002/biot.202000240 32914549
    [Google Scholar]
  32. Wagner J.M. Liu L. Yuan S.F. Venkataraman M.V. Abate A.R. Alper H.S. A comparative analysis of single cell and droplet-based FACS for improving production phenotypes: Riboflavin overproduction in Yarrowia lipolytica. Metab. Eng. 2018 47 346 356 10.1016/j.ymben.2018.04.015 29698778
    [Google Scholar]
  33. Devaraj N.K. Finn M.G. Introduction: Click chemistry. Chem. Rev. 2021 121 12 6697 6698 10.1021/acs.chemrev.1c00469 34157843
    [Google Scholar]
  34. Hamamichi S. Fukuhara T. Hattori N. Immunotoxin screening system: A rapid and direct approach to obtain functional antibodies with internalization capacities. Toxins 2020 12 10 658 10.3390/toxins12100658 33076544
    [Google Scholar]
  35. Han X. Wang Y. Li S. Hu C. Li T. Gu C. Wang K. Shen M. Wang J. Hu J. Wu R. Mu S. Gong F. Chen Q. Gao F. Huang J. Long Y. Luo F. Song S. Long S. Hao Y. Li L. Wu Y. Xu W. Cai X. Gao Q. Zhang G. He C. Deng K. Du L. Nai Y. Wang W. Xie Y. Qu D. Huang A. Tang N. Jin A. A rapid and efficient screening system for neutralizing antibodies and its application for sars-cov-2. Front. Immunol. 2021 12 653189 10.3389/fimmu.2021.653189 33828563
    [Google Scholar]
  36. Jin Z. Wang L. Cao D. Zou S. Chen C. Kang H. Song Q. Wang H. Tang Y. A new method for rapid screening of hybridoma cell clones secreting paired antibodies using sandwich cell surface fluorescence immunosorbent assay. Anal. Chim. Acta 2021 1163 338493 10.1016/j.aca.2021.338493 34024420
    [Google Scholar]
  37. Sun Y. Ban B. Bradbury A. Ansari G.A.S. Blake D.A. Combining yeast display and competitive facs to select rare hapten-specific clones from recombinant antibody libraries. Anal. Chem. 2016 88 18 9181 9189 10.1021/acs.analchem.6b02334 27571429
    [Google Scholar]
  38. von Boehmer L. Liu C. Ackerman S. Gitlin A.D. Wang Q. Gazumyan A. Nussenzweig M.C. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat. Protoc. 2016 11 10 1908 1923 10.1038/nprot.2016.102 27658009
    [Google Scholar]
/content/journals/cchts/10.2174/0113862073352395250211052148
Loading
Rapid Screening and Effective Rabbit-Derived Fab Antibodies Production Based on Yeast Surface Display
Bentham Science Publishers ; https://doi.org/10.2174/0113862073352395250211052148
/content/journals/cchts/10.2174/0113862073352395250211052148
/content/journals/cchts/10.2174/0113862073352395250211052148
Loading

Data & Media loading...


  • Article Type:     Research Article
Keywords: fab antibody ; flow cytometry sorting ; bidirectional promoter ; rapid screening ; Yeast surface display ; brewer's yeast

Most Cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test