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Introduction: The generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques. Methods: Using a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells. Results: 10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor. Conclusions: Our data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach may be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface. [ABSTRACT FROM AUTHOR]

Next-generation approaches for protein sequencing are now emerging that could have the potential to revolutionize the field in proteomics. One such sequencing method involves fluorescence-based imaging of immobilized peptides in which the N-terminal amino acid of a polypeptide is readout sequentially by a series of fluorescently labeled biomolecules. When selectively bound to a specific N-terminal amino acid, the NAAB (N-terminal amino acid binder) affinity reagent identifies the amino acid through its associated fluorescence tag. A key technical challenge in implementing this fluoro-sequencing approach is the need to develop NAAB affinity reagents with the high affinity and selectivity for specific N-terminal amino acids required for this biotechnology application. One approach to develop such a NAAB affinity reagent is to leverage naturally occurring biomolecules that bind amino acids and/or peptides. Here, we describe several candidate biomolecules that could be considered for this purpose and discuss the potential for developability of each. Key points • Next-generation sequencing methods are emerging that could revolutionize proteomics. • Sequential readout of N-terminal amino acids by fluorescent-tagged affinity reagents. • Native peptide/amino acid binders can be engineered into affinity reagents. • Protein size and structure contribute to feasibility of reagent developability. [ABSTRACT FROM AUTHOR]

Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action. [ABSTRACT FROM AUTHOR]

Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research. [ABSTRACT FROM AUTHOR]

Abstract: The development of stable biomolecular scaffolds that can tolerate environmental extremes has considerable potential for industrial and defense-related applications. However, most natural proteins are not sufficiently stable to withstand non-physiological conditions. We have recently engineered the de novo designed Top7 protein to specifically recognize the glycoprotein CD4 by insertion of an eight-residue loop. The engineered variant exhibited remarkable stability under chemical and thermal denaturation conditions. In the present study, far-UV CD spectroscopy and explicit-solvent MD simulations are used to investigate the structural stability of Top7 and the engineered variant under extreme conditions of temperature and pH. Circular dichroism measurements suggest that the engineered variant Top7CB1, like Top7, retains its structure at high temperatures. Changes in CD spectra suggest that there are minor structural rearrangements between neutral and acidic environments for both proteins but that these do not make the proteins less stable at high temperatures. The anti-parallel β-sheet is well conserved within the timescale simulated whereas there is a decrease of helical content when low pH and high-temperature conditions are combined. Concerted alanine mutations along the α-helices of the engineered Top7 variant did not revert this trend when at pH 2 and 400K. The structural resilience of the anti-parallel β-sheet suggests that the protein scaffold can accommodate varying sequences. The robustness of the Top7 scaffold under extreme conditions of pH and temperature and its amenability to production in inexpensive bacterial expression systems reveal great potential for novel biotechnological applications. [Copyright &y& Elsevier]

Histone post-translational modifications are small chemical changes to the histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET-based systems. High-throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet, published studies on their use with histones have been noticeably sparse. Here, we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding. [ABSTRACT FROM AUTHOR]

Previous in vivo investigations have shown that glutathione reductase is one of the sites of action of the dithiocarbamate fungicide tetramethylthiuram disulphide (thiram) in the yeast Saccharomyces cerevisiae. The inactivation of glutathione reductase by thiram has now been demonstrated in vitro. This inactivation was time-dependent and occurred only with the enzyme in the reduced state and in the absence of glutathione. Since the turnover rate of the enzyme with thiram as a substrate was significantly higher than the rate of enzyme inactivation, it was suggested that more than one enzyme-inhibitor complex was involved in the reaction. Arguments supporting a covalent modification of glutathione reductase were further obtained by experiments carried out with [14C]thiram and gel filtration. A kinetic scheme for the inactivation is proposed and the relevance of the in vitro data to previous in vivo studies is discussed taking into consideration current concepts of glutathione reductase inactivation by affinity reagents. [ABSTRACT FROM AUTHOR]

Abstract: With the sequencing of an eukaryotic genome, it is possible to inventory the predicted proteome for proteins that carry one or more Src Homology 3 (SH3) domains. Due to the current ease of cloning and gene synthesis, these short domains can be readily overexpressed and manipulated for the purpose of characterizing their specificity and affinity for peptide ligands, as well as solving the three-dimensional structures of the domains. This information can be used to predict and confirm their cellular interacting partners, in the effort to understand the function of a eukaryotic protein by focusing on its SH3 domain. Finally, capitalizing on our mature understanding about protein–protein interacting modules, like the SH3 domain, it is possible to use directed evolution to enhance or change the specificity and affinity of an SH3 domain for the purpose of creating reagents to be used in biochemical purification or cell perturbation studies. [Copyright &y& Elsevier]

Protein-based binders have become increasingly more attractive candidates for drug and imaging agent development. Such binders could be evolved from a number of different scaffolds, including antibodies, natural protein effectors and unrelated small protein domains of different geometries. While both computational and experimental approaches could be utilized for protein binder engineering, in this review we focus on various computational approaches for protein binder design and demonstrate how experimental selection could be applied to subsequently optimize computationally-designed molecules. Recent studies report a number of designed protein binders with pM affinities and high specificities for their targets. These binders usually characterized with high stability, solubility, and low production cost. Such attractive molecules are bound to become more common in various biotechnological and biomedical applications in the near future., (The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput " all-on-one chip " system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide.

Abstract: For more than 30years, the production of research antibodies has been dominated by hybridoma technologies, while modern recombinant technologies have lagged behind. Here I discuss why this situation must change if we are to generate reliable, comprehensive reagent sets on a genome-wide scale, and I describe how a cultural shift in the research community could revolutionize and modernize the affinity reagent field. In turn, such a revolution would pay huge dividends by closing the gap between basic research and therapeutic development, thus enabling the development of myriad new therapies for unmet medical needs. [Copyright &y& Elsevier]

Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action., Competing Interests: The authors declare no conflict of interest.