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A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes.

ABSTRACT: Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub. The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.

SUBMITTER: Lajoie JM

PROVIDER: S-EPMC7017056 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes.

Lajoie Jason M JM Cho Yong Ku YK Frost Dustin D Bremner Samantha S Li Lingjun L Shusta Eric V EV

Protein engineering, design & selection : PEDS 20191201 5

Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concep ...[more]

PMID: 31769480

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Project description:BackgroundNeutralizing antibody plays a key role in protecting hosts from invasive pathogens and their virulent components. Current high-throughput assays for antibody screening are based on binding activities. However, those antibodies with high affinity may not have neutralizing activities. Subsequent functionality assays are necessary to identify neutralizing antibodies from binders with high affinity to their target antigens, which is laborious and time-consuming. Therefore, a versatile platform that can rapidly identify antibodies with both high binding affinity and neutralizing activity is desired to curb future pandemics like COVID-19.ResultsIn this proof-of-concept study, we adapted Saccharomyces cerevisiae to either display human antibodies on the yeast surface or secrete soluble antibodies into the cultivation supernatant under a controllable 'switch' through different carbon source induced promoters. Initially, an engineered chimeric-bispecific Fab antibody, derived from humanized nanobodies against both Clostridioides difficile toxin A and B (TcdA and TcdB), was successfully expressed either on the yeast cell surface or in the culture medium with intact bioactivity, suggesting the applicability of our system in antibody display and secretion. Next, a combinatorial Fab library was constructed from B cells isolated from a convalescent patient with a high serological neutralizing titer against TcdB. Following three rounds of magnetic bead enrichment and one round of flow cytometry sorting, antibodies against TcdB were enriched efficiently. We then sorted out single binders with high binding affinity and induced them to express soluble antibodies in culture medium. The neutralizing activity of culture supernatant was analyzed using cell-based assay immediately. This way, we rapidly identified two unique neutralizers (out of seven binders) that can neutralize the cytotoxicity of TcdB.ConclusionThe antibody screening platform described here simplifies the neutralizing antibody discovery procedure and will be an attractive alternative for screening functional antibodies against infectious diseases.

Dataset Information

A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes.

Publications

A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes.

Similar Datasets

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