Previously, we have described a miniature protein-based approach to the design of molecules that bind DNA or protein surfaces with high affinity and specificity. In this approach, the small, wellfolded protein avian pancreatic polypeptide acts as a scaffold to present and stabilize an R-helical or PPII-helical recognition epitope. The first miniature protein designed in this way, a molecule called p007, presents the R-helical recognition epitope found on the bZIP protein GCN4 and binds DNA with nanomolar affinity and exceptional specificity. In this work we use alanine-scanning mutagenesis to explore the contributions of 29 p007 residues to DNA affinity, specificity, and secondary structure. Virtually every residue within the p007 R-helix, and most residues within the p007 PPII helix, contribute to both DNA affinity and specificity. These residues include those introduced to make specific and nonspecific DNA contacts, as well as those that complete the miniature protein core. Moreover, there exists a direct correlation between the affinity of a p007 variant for specific DNA and the ability of that variant to select for specific DNA over nonspecific DNA. Although we observe no correlation between R-helicity and affinity, we observe a limited correlation between R-helicity and sequence specificity that emphasizes the role of coupled binding/folding in the function of p007. Our results imply that formation of a highly evolved set of protein‚DNA contacts in the context of a well-packed hydrophobic core, and not the extent of intrinsic R-helical structure, is the primary determinant of p007 function.The development of general strategies for the selective recognition of macromolecular targets remains a major challenge for chemical biology and a fundamental postgenome goal. Molecules capable of tight and selective macromolecular recognition have utility in the validation of potential therapeutic targets and can, in certain cases, function as therapeutics in their own right (1-3). Our laboratory has developed a general approach toward the design of small, well-folded proteins that bind macromolecular targets with high affinity and selectivity (4-11). This approach is often referred to as protein grafting, and the molecules that result are called miniature proteins: miniature because they contain fewer than 40 amino acids and proteins because they often fold cooperatively. In a protein grafting experiment, those residues that comprise a natural R-helical or type II polyproline (PPII)-helical recognition epitope are introduced onto the solvent-exposed R-or PPII-helical face of the small yet stable protein avian pancreatic polypeptide (aPP) 1 (12, 13). Our laboratory has used this procedure, often in combination with directed evolution, to engineer miniature protein ligands with high affinity for a variety of targets including the duplex DNAs recognized by GCN4 and CREB (4, 5) and the Q50K engrailed homeodomain (7), the antiapoptotic proteins Bcl-X L (6) and Bcl-2 (10), the oncoprotein hDM2 (manuscript in prepara...

Biomedical research is evolving to address biological systems as molecular pathways integrated into complex networks. Tools for molecular and cell analysis are also evolving to address the new challenges and opportunities of this approach. Flow cytometry is a versatile analytical platform, capable of high speed quantitative measurements of cells and other particles. These capabilities are being exploited and extended in a range of new applications stemming from opportunities presented by the advances of genomics, proteomics and systems biology, which are in turn impacting clinical diagnosis, vaccine development and drug discovery. In this review, we highlight some of these advances and consider the future evolution of flow cytometry technology.

Clones from phage display libraries are generally selected by a number of rounds of panning and regrowth, followed by primary screening to identify hits and secondary characterization to identify clones with optimal affinity and specificity. Because functional screening for binding or other activity can be material-, time-, and labor-intensive, sequencing is often used to identify the emergence of a consensus sequence prior functional characterization. However, the consensus sequence is not always the optimal one because factors such as phage growth rates, nonspecific binding, and other selection pressures can bias the selection process. To improve function-based phage display library screening and characterization, we developed a multiplexed approach employing optically-encoded microsphere arrays and flow cytometry. We show that capture of phage from crude culture supernatants enables the efficient screening of binding activity and the evaluation of binding avidity. The approach uses small volumes and a homogeneous no-wash format that minimizes reagent consumption and sample handling. The use of opticallyencoded microspheres allows many phage to be screened simultaneously, greatly increasing throughput. This approach is flexible, supporting primary and secondary screening for a range of functional assays, and scalable, potentially supporting the screening of thousands to hundreds of thousands of clones per hour. ' 2007

The majority of mammalian proteins are glycosylated, with the glycans serving to modulate a wide range of biological activities. Variations in protein glycosylation can have dramatic effects on protein stability, immunogenicity, antibody effector function, pharmacological safety and potency, as well as serum half-life. The glycosylation of therapeutic biologicals is a critical quality attribute (CQA) that must be carefully monitored to ensure batch-to-batch consistency. Notably, many factors can affect the composition of the glycans during glycoprotein production, and variations in glycosylation are among the leading causes of pharmaceutical batch rejection. Currently, the characterization of protein glycosylation relies heavily on methods that employ chromatography and/or mass spectrometry, which require a high level of expertise, are time-consuming and costly and, because they are challenging to implement during in-process biologics production or during in vitro glycan modification, are generally performed only post-production. Here we report a simplified approach to assist in monitoring glycosylation features during glycoprotein engineering, that employs flow cytometry using fluorescent microspheres chemically coupled to high-specificity glycan binding reagents. In our GlycoSense method, a range of carbohydrate-sensing microspheres with distinct optical properties may be combined into a multiplex suspension array capable of detecting multiple orthogonal glycosylation features simultaneously, using commonplace instrumentation, without the need for glycan release. The GlycoSense method is not intended to replace more detailed post-production glycan profiling, but instead, to complement them by potentially providing a cost-effective, rapid, yet robust method for use at-line as a process analytic technology (PAT) in a biopharmaceutical workflow or at the research bench. The growing interest in using in vitro glycoengineering to generate glycoproteins with well-defined glycosylation, provides motivation to demonstrate the capabilities of the GlycoSense method, which we apply here to monitor changes in the protein glycosylation pattern (GlycoPrint) during the in vitro enzymatic modification of the glycans in model glycoproteins.

Glycans have distinct properties that make them appealing as disease biomarker targets, and novel highly specific reagents are essential to overcome current limitations in the discovery and exploitation of disease‐related glycans.Lectenz® Bio is engineering glycan‐processing enzymes into catalytically inactive, high affinity glycan binding reagents with tunable specificities. These novel lectin‐like, enzyme‐derived reagents called Lectenz® are being developed for a variety of glycan detection and enrichment applications including affinity chromatography, Western blot, and immunohistochemistry. The conversion of such enzymes into affinity reagents is facilitated by computationally‐guided directed evolution. Using site‐directed mutagenesis and yeast display selection of a site‐saturation mutagenesis library, multiple Lectenz® candidates can be identified.Here we present two novel sialic acid recognizing Lectenz® engineered from a sialidase enzyme: 1) the pan‐specific sialic acid reagent, Sia‐PS1 Lectenz®, which recognizes sialic acid in a linkage independent manner; and 2) the Sia‐3S1 Lectenz®, which is specific for α2,3‐linked sialoglycans. The data demonstrate that the Sia‐PS1 Lectenz® reagent recognizes glycans terminating in α2,3‐, α2,6‐, and α2,8‐linked sialic acid sequences, but not Gal‐terminating sequences. With the Sia‐3S1 Lectenz®, we demonstrate binding to Neu5Acα2,3Galβ1,4Glc, but not Neu5Acα2,6 Galβ1,4Glc or Galβ1,4Glc.Support or Funding InformationNational Institutes of Health (R41GM113351)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.