A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping

Grzeschik, Julius; Hinz, Steffen C.; Könning, Doreen; Pirzer, Thomas; Becker, Stefan; Zielonka, Stefan; Kolmar, Harald

doi:10.1002/biot.201600454

Cited by 28 publications

(19 citation statements)

References 20 publications

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“…Simultaneous expression of heavy- and light-chains is enabled via the introduction of the picornaviral 2A peptide, mediating ribosomal skipping and therefore the lack of peptide bond formation between Aga2p which is fused to the heavy chain and the light chain. In contrast to internal ribosome entry sites, ribosomal skipping results in the translation of equal amounts of each protein and it has been shown previously that the 2A peptide is a versatile tool for yeast surface display [ 21 , 33 ]. Similarly to the two-directional display system, signature sequences were constituents of sequence-encoding antibody constant regions or signal sequences and Bsa I sites were designed to be removed during restriction-ligation reaction, allowing for seamless one-step cloning of the final library display vector (pExp_bicis).…”

Section: Resultsmentioning

confidence: 99%

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A novel one-step approach for the construction of yeast surface display Fab antibody libraries

et al. 2018

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BackgroundYeast 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.ResultsWithin 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0853-z) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

“…In the bicistronic system (bicis), Fab display is mediated by ribosomal skipping (Fig. 1 b) [ 21 , 22 ]. We show that large antibody libraries with more than 10 8 unique clones can readily be constructed by applying the herein presented approach.…”

Section: Introductionmentioning

confidence: 99%

A novel one-step approach for the construction of yeast surface display Fab antibody libraries

et al. 2018

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“…To avoid both possible steric hindrance by secondary fluorescence antibody, and the time-consuming and expensive tagging process of yeast expression, we decided to use an alternative yeast display construct in which a T2A-EGFP sequence fused to the C terminus of the displayed protein allows intracellular EGFP expression to be correlated with display level of the surface proteins ( Fig. 2B) (19). We observed that the EGFP expression level indicates a similar expression profile for PcTx1.…”

Section: Resultsmentioning

confidence: 99%

A cell–cell interaction format for selection of high-affinity antibodies to membrane proteins

Yang

Wan

Tao

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Generating and improving antibodies and peptides that bind specifically to membrane protein targets such as ion channels and G protein-coupled receptors (GPCRs) can be challenging using established selection methods. Current strategies are often limited by difficulties in the presentation of the antigen or the efficiency of the selection process. Here, we report a method for obtaining antibodies specific for whole cell membrane-associated antigens which combines a cell–cell interaction format based on yeast display technology with fluorescence-activated cell sorting of dual fluorescent complexes. Using this method, we were able to direct the affinity maturation of an antagonist antibody specific for the proton-gated ion channel ASIC1a and showed that both the affinity and potency were improved. We were also able to use this method to do kinetic selections to generate clones with better dissociation profiles. In addition, this method was employed successfully to handle the difficult problem of selecting antibodies specific to a GPCR target, the mu-opioid receptor.

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“…Ribosomal skipping (T2A peptide) results in the secretion of the mIL-3 protein, carrying a secretion signal (app8 31 ) www.nature.com/scientificreports www.nature.com/scientificreports/ (Supplementary Table S1). Simultaneously, mCherry fluorescent protein accumulates intracellularly in a correlation to the expression rate of the cytokine, as demonstrated by Grzeschik et al 32 , and thus serves as an expression control.…”

Section: Activation Of Ba/f3-cis-d2egfp Reporter Cells Using Mil-3 Comentioning

confidence: 85%

FACS-Based Functional Protein Screening via Microfluidic Co-encapsulation of Yeast Secretor and Mammalian Reporter Cells

Yanakieva

Elter

Bratsch

et al. 2020

Sci Rep

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in this study, we present a straightforward approach for functional cell-based screening by coencapsulation of secretor yeast cells and reporter mammalian cells in millions of individual agarosecontaining microdroplets. our system is compatible with ultra-high-throughput selection utilizing standard fluorescence-activated cell sorters (FACS) without need of extensive adaptation and optimization. In a model study we co-encapsulated murine interleukin 3 (mIL-3)-secreting S. cerevisiae cells with murine Ba/F3 reporter cells, which express green fluorescent protein (GFP) upon stimulation with mIL-3, and could observe specific and robust induction of fluorescence signal compared to a control with yeast cells secreting a non-functional mIL-3 mutant. We demonstrate the successful enrichment of activating mIL-3 wt-secreting yeast cells from a 1:10,000 dilution in cells expressing the inactive cytokine variant by two consecutive cycles of co-encapsulation and fAcS. this indicates the suitability of the presented strategy for functional screening of high-diversity yeast-based libraries and demonstrates its potential for the efficient isolation of clones secreting bioactive recombinant proteins. The importance of biopharmaceuticals (biologics) in medicine is increasing at a fast pace and the biologics market is predicted to reach nearly 400 billion USD/year by 2025 1. Frequently applied biologics comprise substances such as cytokines, monoclonal antibodies, hormones, soluble receptors, recombinant DNAs, enzymes, and synthetic vaccines. While biologics in targeted therapies often demonstrate remarkable safety and specificity, especially in case of autoimmune diseases 2 and cancer 3 , the discovery of novel molecules and the necessary functional validation still represent a bottleneck in the development of novel biopharmaceuticals. To overcome these shortcomings, powerful display technologies such as phage display 4 and yeast surface display 5 have been developed which allow for the isolation of specific high-affinity molecules and respective genes from complex variant libraries. However, identified binders frequently show poor physiological activity in a biological context. Thus, extensive secondary functional screens are necessary for identification of hit molecules with the desired functional activity. Furthermore, those screens require elaborate subcloning of the surface-displayed hits into soluble expression formats and outcoming clones frequently demand further optimization of physicochemical and pharmacokinetic properties 6. Consequently, implementing functional assays and phenotypic screens in an earlier selection phase appears highly beneficial for the discovery of new potent biologic drugs or even first-in-class medicines with novel molecular mechanisms of action 7. In this context, a major limitation is represented by the relatively low throughput of classical phenotypic screens, falling far behind the performance of high-diversity library-based approaches resting on affinity-driven selection protocols 8. Complex p...

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A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping

Cited by 28 publications

References 20 publications

A novel one-step approach for the construction of yeast surface display Fab antibody libraries

A novel one-step approach for the construction of yeast surface display Fab antibody libraries

A cell–cell interaction format for selection of high-affinity antibodies to membrane proteins

FACS-Based Functional Protein Screening via Microfluidic Co-encapsulation of Yeast Secretor and Mammalian Reporter Cells

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