Affinity maturation of Fab antibody fragments by fluorescent‐activated cell sorting of yeast‐displayed libraries

Beucken, Twan van den; Pieters, Henk; Steukers, Mieke; Vaart, Marcel van der; Ladner, Robert C.; Hoogenboom, Hennie R.; Hufton, Simon E.

doi:10.1016/s0014-5793(03)00602-1

Cited by 94 publications

(52 citation statements)

References 30 publications

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“…Our new system will offer a method to select antibodies against antigens that need to be expressed in eukaryotes. Other applications of YS2H may include expression of heterodimeric proteins, as demonstrated by similar platforms for expression of heterodimeric mammalian proteins such as major histocompatibility complex II ␣ and ␤ subunits (29) and antibodies in Fab format (30). The use of our system to quantify and discover protein-protein interactions is not necessarily limited to the study of secretory proteins, because many proteins in nonsecretory cellular compartments or cytosol will maintain native conformations and interactions.…”

Section: Discussionmentioning

confidence: 99%

Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

Kang

Chen

et al. 2009

Journal of Biological Chemistry

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A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed "yeast surface two-hybrid (YS2H)," to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two ␣-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pM to 10 M. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pM to 100 M. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chainonly antibodies against botulinum neurotoxin.Protein-protein interactions are essential to virtually every cellular process, and their understanding is of great interest in basic science as well as in the development of effective therapeutics. Existing techniques to detect and screen pairs of interacting proteins in vivo include the yeast two-hybrid system (1) and protein-fragment complementation assay (PCA) 2 (2-6),where the association of two interacting proteins either turns on a target gene that is necessary for cell survival or leads to the reconstitution of enzymes or green fluorescence protein (GFP) or its variants. The application of protein-protein interactions that are probed with yeast two-hybrid and PCA has been focused mainly on the interactions occurring in the nucleus or cytosol. To study interactions among secretory proteins and membrane-associated proteins, a variant of yeast two-hybrid has been developed for detecting protein-protein interactions occurring in the secretory pathway (7,8). However, most existing methods are designed to map connectivity information for pairwise interactions and are not suitable for measuring the affinity between two interacting proteins, comparing interaction strength of different pairs, or ranking multiple binders to the interaction "hub" according to their binding affinity. Quantitative estimation of protein-protein interactions in vivo will req...

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Section: Discussionmentioning

confidence: 99%

Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

Kang

Chen

et al. 2009

Journal of Biological Chemistry

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“…scFv clones with improved properties could be efficiently isolated from the library through the incubation of scFv-displaying yeast library with fluorescently labeled antigen and the subsequent screening by fluorescently activated cell sorting (FACS) [7]. Furthermore, antibodies to botulinum neurotoxins, carcinoembryonic antigen, CD3 diphtheria toxin, lysozyme, and streptavidin have been improved or developed using the similar strategies based on arming technology [96,97,98,99,100,101]. Thus, arming technology is an attractive methodology for the isolation of antibodies with high affinity and other specific biological functions.…”

Section: Protein Engineering and Directed Evolution By Arming Techmentioning

confidence: 99%

Arming Technology in Yeast—Novel Strategy for Whole-cell Biocatalyst and Protein Engineering

Kuroda

Ueda

2013

Biomolecules

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Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called “arming technology”, can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach.

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“…Therefore several display strategies have been developed to select higher-affinity antibodies from libraries expressing mutated variants of Fabs or scFvs via phage display, E.coli and yeast surface display and ribosome display [65,67,71,72,73]. Usually, in vitro affinity maturation starts with a lead antibody with medium affinity that is processed in (thioredoxin and glutathione/glutaredoxin pathways) [82].…”

Section: Expression and Purification Strate-gies Of Recombinant Antibmentioning

confidence: 99%

Considerations on Antibody-Phage Display Methodology

Conrad

Scheller²

2005

CCHTS

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For almost 15 years phage display is being used for the selection of specific antigen-binders from artificial libraries of single chain antibodies. Filamentous phages have been developed in a way to express foreign proteins on the surface and at the same time carrying the genetic information of the surface expressed molecule within the phage capsid. This property guarantees the coupling of phenotype and genotype during phage amplification and affinity selection. The possibility to generate large antibody libraries and the simplified antibody-backbone of a single chain antibody has made antibody-phage display to a powerful tool for the development of new therapeutics against various human diseases. In this review we discuss the general principles and latest developments and applications in antibody phage display technology.

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Affinity maturation of Fab antibody fragments by fluorescent‐activated cell sorting of yeast‐displayed libraries

Cited by 94 publications

References 30 publications

Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

Arming Technology in Yeast—Novel Strategy for Whole-cell Biocatalyst and Protein Engineering

Considerations on Antibody-Phage Display Methodology

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