A novel one-step approach for the construction of yeast surface display Fab antibody libraries
Abstract Background Yeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids i...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2018-01-01
|
| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12934-017-0853-z |
| _version_ | 1818274890433691648 |
|---|---|
| author | Simon Rosowski Stefan Becker Lars Toleikis Bernhard Valldorf Julius Grzeschik Deniz Demir Iris Willenbücher Ramona Gaa Harald Kolmar Stefan Zielonka Simon Krah |
| author_facet | Simon Rosowski Stefan Becker Lars Toleikis Bernhard Valldorf Julius Grzeschik Deniz Demir Iris Willenbücher Ramona Gaa Harald Kolmar Stefan Zielonka Simon Krah |
| author_sort | Simon Rosowski |
| collection | DOAJ |
| description | Abstract Background Yeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. Results Within this study, we aimed at implementing a focused Golden Gate Cloning approach for the generation of YSD libraries. For this, antibodies heavy and light chains were encoded on one single plasmid. Fab display on yeast cells was either mediated by a two-directional promoter system (2dir) or by ribosomal skipping (bicis). The general applicability of this methodology was proven by the functional display of a therapeutic antibody. Subsequently, we constructed large antibody libraries with heavy chain diversities derived from CEACAM5 immunized animals in combination with a common light chain. Target-specific antibodies from both display systems were readily obtained after three rounds of fluorescence activated cell sorting. Isolated variants exhibited high affinities in the nanomolar and subnanomolar range as well as appropriate biophysical properties. Conclusion We demonstrated that Golden Gate Cloning appears to be a valid tool for the generation of large yeast surface display antibody Fab libraries. This procedure simplifies the hit discovery process of antibodies from immune repertoires. |
| first_indexed | 2024-12-12T22:21:03Z |
| format | Article |
| id | doaj.art-0026bb751c724f14a378aa178f21a5dd |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-12T22:21:03Z |
| publishDate | 2018-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-0026bb751c724f14a378aa178f21a5dd2022-12-22T00:09:55ZengBMCMicrobial Cell Factories1475-28592018-01-0117111110.1186/s12934-017-0853-zA novel one-step approach for the construction of yeast surface display Fab antibody librariesSimon Rosowski0Stefan Becker1Lars Toleikis2Bernhard Valldorf3Julius Grzeschik4Deniz Demir5Iris Willenbücher6Ramona Gaa7Harald Kolmar8Stefan Zielonka9Simon Krah10Institute for Organic Chemistry and Biochemistry, Technische Universität DarmstadtProtein Engineering and Antibody Technologies, Merck KGaAProtein Engineering and Antibody Technologies, Merck KGaAChemical and Pharmaceutical Development, Merck KGaAInstitute for Organic Chemistry and Biochemistry, Technische Universität DarmstadtProtein Engineering and Antibody Technologies, Merck KGaAProtein Engineering and Antibody Technologies, Merck KGaAProtein Engineering and Antibody Technologies, Merck KGaAInstitute for Organic Chemistry and Biochemistry, Technische Universität DarmstadtProtein Engineering and Antibody Technologies, Merck KGaAProtein Engineering and Antibody Technologies, Merck KGaAAbstract Background Yeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. Results Within this study, we aimed at implementing a focused Golden Gate Cloning approach for the generation of YSD libraries. For this, antibodies heavy and light chains were encoded on one single plasmid. Fab display on yeast cells was either mediated by a two-directional promoter system (2dir) or by ribosomal skipping (bicis). The general applicability of this methodology was proven by the functional display of a therapeutic antibody. Subsequently, we constructed large antibody libraries with heavy chain diversities derived from CEACAM5 immunized animals in combination with a common light chain. Target-specific antibodies from both display systems were readily obtained after three rounds of fluorescence activated cell sorting. Isolated variants exhibited high affinities in the nanomolar and subnanomolar range as well as appropriate biophysical properties. Conclusion We demonstrated that Golden Gate Cloning appears to be a valid tool for the generation of large yeast surface display antibody Fab libraries. This procedure simplifies the hit discovery process of antibodies from immune repertoires.http://link.springer.com/article/10.1186/s12934-017-0853-zGolden Gate CloningYeast surface displayFab fragmentAntibody discovery |
| spellingShingle | Simon Rosowski Stefan Becker Lars Toleikis Bernhard Valldorf Julius Grzeschik Deniz Demir Iris Willenbücher Ramona Gaa Harald Kolmar Stefan Zielonka Simon Krah A novel one-step approach for the construction of yeast surface display Fab antibody libraries Microbial Cell Factories Golden Gate Cloning Yeast surface display Fab fragment Antibody discovery |
| title | A novel one-step approach for the construction of yeast surface display Fab antibody libraries |
| title_full | A novel one-step approach for the construction of yeast surface display Fab antibody libraries |
| title_fullStr | A novel one-step approach for the construction of yeast surface display Fab antibody libraries |
| title_full_unstemmed | A novel one-step approach for the construction of yeast surface display Fab antibody libraries |
| title_short | A novel one-step approach for the construction of yeast surface display Fab antibody libraries |
| title_sort | novel one step approach for the construction of yeast surface display fab antibody libraries |
| topic | Golden Gate Cloning Yeast surface display Fab fragment Antibody discovery |
| url | http://link.springer.com/article/10.1186/s12934-017-0853-z |
| work_keys_str_mv | AT simonrosowski anovelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT stefanbecker anovelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT larstoleikis anovelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT bernhardvalldorf anovelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT juliusgrzeschik anovelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT denizdemir anovelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT iriswillenbucher anovelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT ramonagaa anovelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT haraldkolmar anovelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT stefanzielonka anovelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT simonkrah anovelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT simonrosowski novelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT stefanbecker novelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT larstoleikis novelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT bernhardvalldorf novelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT juliusgrzeschik novelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT denizdemir novelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT iriswillenbucher novelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT ramonagaa novelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT haraldkolmar novelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT stefanzielonka novelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries AT simonkrah novelonestepapproachfortheconstructionofyeastsurfacedisplayfabantibodylibraries |