Direct selection of functional fluorescent-protein antibody fusions by yeast display

PLOS ONE RESEARCH ARTICLE Direct selection of functional fluorescentprotein antibody fusions by yeast display Nileena Velappan1☯*, Fortunato Ferrara ID2☯, Sara D’Angelo2, Devin Close3, Leslie Naranjo2, Madeline R. Bolding ID1, Sarah C. Mozden ID1, Camille B. Troup4, Donna K. McCullough5, Analyssa Gomez1, Marijo Kedge1, Andrew R. M. Bradbury ID2* 1 Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, United States of America, 2 Specifica Inc., Santa Fe, NM, United States of America, 3 Arup Laboratories, Salt Lake City, UT, United States of America, 4 Carterra Inc., Salt Lake City, UT, United States of America, 5 Microbiology Department, University of Tennessee, Knoxville, TN, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Velappan N, Ferrara F, D’Angelo S, Close D, Naranjo L, Bolding MR, et al. (2023) Direct selection of functional fluorescent-protein antibody fusions by yeast display. PLoS ONE 18(2): e0280930. https://doi.org/10.1371/journal. pone.0280930 Editor: Jinny L. Liu, Naval Research Laboratory, UNITED STATES Received: June 14, 2022 ☯ These authors contributed equally to this work. * (ARMB); (NV) Abstract Antibodies are important reagents for research, diagnostics, and therapeutics. Many examples of chimeric proteins combining the specific target recognition of antibodies with complementing functionalities such as fluorescence, toxicity or enzymatic activity have been described. However, antibodies selected solely on the basis of their binding specificities are not necessarily ideal candidates for the construction of chimeras. Here, we describe a high throughput method based on yeast display to directly select antibodies most suitable for conversion to fluorescent chimera. A library of scFv binders was converted to a fluorescent chimeric form, by cloning thermal green protein into the linker between VH and VL, and directly selecting for both binding and fluorescent functionality. This allowed us to directly identify antibodies functional in the single chain TGP format, that manifest higher protein expression, easier protein purification, and one-step binding assays. Accepted: January 11, 2023 Published: February 24, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0280930 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction Protein chimeras are recombinant proteins created by linking the genes of different proteins, with the intention of combining their functional properties [1]. These fusion proteins often combine the binding activity of one protein with the enzymatic activity, toxicity, fluorescence, improved solubility, expression or folding of a second protein. Antibodies are most used to confer binding activity, and scFvs the most commonly used format for antibody chimeras. These reprise the antigen-binding properties of full-length antibodies in a smaller, single gene construct [2–4] and are usually selected from immune or naïve libraries using phage display [5–12]. The properties of selected antibodies can be significantly enhanced by combining phage and yeast display, which provides greater control over selection parameters and improves the affinity and diversity of selected scFvs [13–17]. Engineered fluorescent proteins play major roles in biological research and are extensively used to study gene expression, protein function, tracking, and protein-protein interaction [18]. Chimeras in which antibody fragments are combined with fluorescent proteins provide PLOS ONE | https://doi.org/10.1371/journal.pone.0280930 February 24, 2023 1 / 22 PLOS ONE Funding: This work was supported by National Institutes of Health Grant P50GM085273 Foundation for the National Institutes of Health [P50GM085273]; Los Alamos National Laboratory’s Laboratory Directed Research & Development grant 20220108ER. Specifica Inc, a Q2 Solutions Company, provided support in the form of salaries for authors FF, SD, LN and ARMB, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing interests: FF, SD, LN and ARMB are employed at Specifica Inc, a Q2 Solutions Company. CBT is employed at Carterra. The commercial affiliations do not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors declare no conflict of interest. High throughput antibody-chimera selection advantages during the evaluation of protein expression, folding, purification and the detection of binding. While antibodies have been combined with fluorescent proteins in many different formats [19–26], there is an inherent complexity involved in the creation of such chimeras since antibodies are secreted proteins and fluorescent proteins are expressed in the cytoplasm. Consequently, many published chimeras have very low expression levels [20], a problem that has been addressed with the split GFP system [27], in which a short tag derived from GFP, rather than GFP itself, is fused to a scFv [28], or by using particularly stable scFvs [22] or nanobodies [29]. An unexpected partial solution to this problem was the finding that fluorescent proteins could be used as linkers between VH and VL [30,31], with the resultant chimeras often better expressed than the parental scFv [32]. We named these chimeric proteins scFPs (single chain fluorescent proteins) [32], according to the fluorescent protein used–scTGP, when thermal green protein [33] was used, and scGFP, when superfolder GFP [34] was used. Unfortunately, while some poorly expressed scFvs were expressed far better as scFPs, not all scFPs were equally well-expressed. As it was impossible to predict a priori which scFvs would have their expressions improved, we converted highly specific scFvs to scTGPs and scGFPs, one at a time, to evaluate their functionality. When expressed well, the intrinsically fluorescent scFPs were effectively displayed on yeast and functioned similarly to the corresponding scFvs with equivalent affinities. Their intrinsic fluorescence allowed straightforward assessment of their expression levels and purification. scFPs do not necessarily need to be secreted and can sometimes be effectively expressed in the bacterial cytoplasm, sometimes at h (...truncated)