The authors would like to acknowledge the support of the U.S. Army Research Laboratory (ARL) Postdoctoral Fellowship Program, administered by the Oak Ridge Associated Universities through a contract with ARL, through the appointment of Dr. Justin P. Jahnke. Remaining funding was provided by the Sensors and Electron Devices Directorate at ARL. The authors would also like to thank the Daugherty Lab at UCSB for sharing the bacterial display library and information for cloning the YPet Mona reagent, Dr. Bryn Adams for support in cloning the YPet Mona reagent at ARL, Dr. Joshua Kogot for his initial work on and training in biopanning of bacterial display libraries, Alena Calm of Edgewood Chemical and Biological Center for sharing plasmids used to express and purify abrax and rivax proteins, Brandi Dorsey for technical support for isolation of PA binding peptides, Qin Guo for technical support of preliminary experiments for isolation of abrax binding peptides, and Dr. Jessica Terrell for helpful discussions regarding this manuscript.

The authors have nothing to disclose

Biopanning bacterial display libraries has been a successful approach to the isolation of affinity reagents, and peptides offer a useful alternative to antibodies for recognition when specific criteria are required, such as stability in extreme environments. Biopanning of these libraries has been streamlined using the autoMCS methods described here, and a commercially available autoMCS platform extends this technology to a much wider audience. In comparison to traditional alternating MCS/FACS sorting methods for bacterial display libraries, autoMCS methods are less expensive because they do not require investment in a more expensive FACS instrument capable of sorting and isolating the bound cells, rather than the type of flow cytometer that is used here, which is only capable of analysis 14. The critical steps for successful biopanning by autoMCS include separation program selection, negative sorting against potential cross-reactants to reduce false positives, monitoring library enrichment using FACS to determine when to stop biopanning, and smart analysis of the candidate pool to avoid cumbersome screening of hundreds of candidates.
The examples described herein demonstrate successful biopanning of the chosen bacterial display library for two separate targets, PA and abrax, although with slightly different analysis strategies due to differences in the peptide sequences for each pool of candidates after four rounds of biopanning. PA was the more straightforward case, with sequence analysis demonstrating clear trends from the peptide sequences that repeated at least once in 144 colonies. This alone was enough to determine a consensus sequence for the PA target, and those colonies that contained the consensus or a similar sequence were the best candidates in terms of the ratio of binding to PA versus binding to the streptavidin negative control. However, this will not always be the case. For the abrax target, sequencing 100 colonies led to five repeating sequences but the trend in these sequences was less clear, other than a tendency to contain aromatic amino acid residues and aspartic acid or asparagine. The predicted &#34;consensus&#34; from the repeating sequences only could have been produced and tested for affinity to abrax, but since no parent sequences contained that exact consensus sequence, we returned to the list of sequenced colonies and observed that other candidates that had more trends in common with each other. In fact, two colonies contained a similar sequence to the &#34;consensus&#34; from the repeats alone, (FWDTWF instead of FWAWF), which had the same trend of tryptophan, phenylalanine, and aspartic acid residues but with different spacing. One of these peptides was a repeating sequence, one was not. These two sequences, along with a third sequence containing a similar variant, were among the top 5 binders tested in terms of affinity for abrax. The top binder overall, AX-A05, also displayed similar trends. The results for abrax demonstrate that an approach which alternates several times between sequence analysis and affinity and specificity analysis will sometimes be required, depending on the chosen target. The results for the abrax target also demonstrate the level of specificity that can be achieved over a very similar protein (rivax 1,15).
The autoMCS programs selected for our studies were Posselds and Posseld, which are both for positive selection of labeled target cells with low frequency, less than 5% of the initial population. In both cases, cells pass through two magnetic columns. In the case of the Posselds program, however, cells pass more slowly through the first column to increase exposure time 41. In addition to the program descriptions given by the manufacturer of the commercial autoMCS device (see Materials Table) 41, these programs were chosen after an initial comparison of several potential programs. Each program&#39;s ability to avoid pulling out false positives from a homogenous culture of negative control cells, and to successfully isolate a known binder to a target of interest from a mixed culture containing negative control cells spiked with a decreasing percentage of the known binder, were compared. If significantly deviating from the applications described here, a similar comparison could be helpful in program selection for the new application. However, previously published results comparing these two programs (Posseld and Posselds) for isolation of peptide affinity reagents for PA showed that using either of these programs for all four rounds of biopanning led to isolation of peptides with similar affinity and specificity for PA and determination of the WXCFTC consensus. The main differences were that Posseld, with its faster flow rate, sorted more rapidly and binding affinity for the target was therefore higher after only two rounds of biopanning, while using Posselds led to more unique sequences after four rounds of biopanning 14. Learning from this, the strategy was changed slightly when biopanning for abrax binding peptides to incorporate both benefits: Posselds was used for Round 1 to allow more exposure time during this critical step where diversity is highest, but then the program was switched to Posseld for faster isolation of the highest affinity binders during Rounds 2-4 1,15. This appeared to be ideal for abrax, especially since the nMFI and percent binding were both higher for abrax sorting round 4 as compared to PA sorting round 4 with either program (Figure 2 and Figure 3 and Sarkes et al. 2015 14), but a direct comparison using one strategy versus the other with the same target would be required for a more conclusive comparison. Which program is best for a particular application may depend on the individual target, the sorting library used, and the level of peptide expression that is achieved with any change to the conditions described here.
Not discussed here are potential results from biopanning with abiotic materials, but we have also used the same bacterial display library for biopanning against bulk aluminum 2. This study was not performed using autoMCS, but similar studies could be accomplished using autoMCS if the material is available on, or could be coated on or conjugated to, a magnetic bead. In the bulk aluminum study, individual amino acid analysis and secondary structure modeling was helpful in understanding what was driving the binding affinity of the isolated peptides, since no consensus sequence was determined 2. Even with biological targets, this may be required to understand what is driving the binding affinity for some targets, which is why the spreadsheet generated from the &#34;eCPX_Sequencing&#34; macro includes a tab with individual residue frequency analysis. If no repeating sequences are seen in addition to the lack of a consensus, and residue frequency shows no pattern, however, other types of analysis may be required. Additionally, further sorting rounds may be necessary to avoid screening a much larger number of candidates for down-selection of those with sufficient affinity to the desired target.
With the increasing availability of next generation sequencing, a more thorough study could be performed to determine the most promising candidates before testing affinity and to determine the extent of enrichment after each round of biopanning. However, sequences moving on to the binding analysis step may need to be re-created by cloning, if they are not of high enough frequency to easily isolate with routine colony sequencing of tens or hundreds of colonies. As this technology advances, becoming less expensive and more routine, and especially with an option for colony isolation, it will likely be the best way to analyze biopanning data. It may lead to elucidation of the way individual sequences, and the entire library, evolve. Additionally, the bacteria in the sorting library may mutate over time, which could bias sorting towards cells with faster growth rates or bacteria that overexpress genomic proteins able to bind a material even without display scaffold and peptide expression, for instance. For that reason, once promising candidates emerge, it is worth isolating the plasmid DNA, retransforming fresh E. coli, and confirming peptide binding via FACS. If planning to use the peptide in a cell-free format, affinity should also be assessed for the free peptide. For example, peptide affinity reagents discovered through bacterial display for the target SEB were synthetically produced off-cell and analyzed by more traditional methods such as enzyme-linked immunosorbant assay (ELISA), and on-cell (via FACS) and off-cell (via ELISA) affinity were compared using the Kds determined by both methods 7.
Although the strength of the biotin-streptavidin interaction makes it an ideal strategy for capture of protein target and bound peptides, this procedure can be modified for other capture strategies. Direct coupling of protein to magnetic beads, such as epoxy and carboxylic acid beads, has been successful and removes a binding step. However, direct attachment may hinder potential binding sites in a specific or non-specific manner, depending on attachment strategy. An additional advantage to using streptavidin beads is that less stable protein targets can be freshly biotinylated in 30 min or stored frozen, if needed, while the beads themselves tend to require longer incubation with the protein for cross-linking and are generally stored at 2 - 8&#176;C. The suggested starting concentration of biotinylated protein target here, 600 nM, has been successful for multiple targets, but it may be possible to isolate peptide capture reagents with increased affinity if this concentration is reduced. Additionally, this method can be extended to and modified for other bacterial display libraries and other organisms. For example, these steps can be performed in the absence of oxygen for use with anaerobes, or in other environments for use with extremophiles to isolate peptides with unique properties. The peptides and/or consensus determined for a particular target of interest can be potentially used on-cell as a living material, or produced synthetically off-cell. Synthetically produced peptides can be further matured for the development of even higher affinity and more robust Protein Catalyzed Capture (PCC) agents for use in sensors, or for other applications where antibodies would typically be used for binding or recognition 38,55. Molecular modeling can also be used to help determine binding location of the peptide with its target, as was recently performed for the abrax binding peptides and consensus listed in Figure 5C 1. Overall, biopanning bacterial display libraries for peptide affinity reagents is a fast, straightforward, and powerful alternative to the production of antibodies for recognition and detection of protein targets, and this semi-automated biopanning approach has produced reliable results with far reaching applications.

Biopanning is a proven technique for affinity reagent discovery, with bacterial display libraries a convenient source for peptide affinity reagents 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. Similarly to phage display 25,26 and yeast display 27,28, the peptides are exposed on the cell surface and are available to bind to both biotic and abiotic targets, with these interactions driven by both the peptide backbone and the unique properties of the amino acid side-chains 29. In contrast to phage display, the bacteria themselves are self-replicating and contain all genetic material required for display without elution of bound phage and reinfection of cells. In contrast to yeast display, bacterial display is faster due to the rapid (approximately 20 min 30) doubling time of Escherichia coli. The resulting peptide affinity reagents can be produced off-cell for use in a variety of sensing platforms 16,17,26 and have potential for use as living materials when displayed on-cell 2,31,32. As compared to monoclonal antibodies for recognition of specific targets, peptides are much smaller and more flexible, and peptide display libraries can be easily manipulated at the DNA level to avoid specific amino acids, such as cysteine 33. Peptides are increasingly exploited for therapeutics 34,35,36 and other applications where antibodies would typically be utilized due to their ease of discovery, reproducible synthetic (off-cell) production, and superior maintenance of performance in extreme environments, providing a functional reagent even after exposure to high temperature or long-term storage without refrigeration 37,38. The ability to overcome the cold chain for storage of reagents is of particular interest to the Department of Defense, for instance, which must operate in extreme environments. Thermal stability was therefore a desirable feature for the affinity reagents recognizing the chosen targets given as examples in this manuscript.
Recently, biopanning bacterial display libraries has become even more straightforward and reliable with the use of semi-automated magnetic-activated cell sorting (MACS or MCS) methods 9,14,39. These methods have been demonstrated for biological targets using several similar sorting platforms with different advantages, including a commercially available automated magnetic-activated cell sorting (autoMACS or autoMCS) instrument (see table of materials) 1,14,15 and more specialized devices which enclose the sample in a disposable cartridge 7,9 or chip 39, a valuable specification for use with organisms or toxins that are Biosafety Level 2 (BSL-2) or higher7. These semi-automated methods could be extended to sort for peptides able to bind to abiotic materials if the materials are magnetic or have the ability to be coated onto a magnetic bead, and for use with different organisms (such as anaerobic bacteria) if the sorting and growth conditions are altered. In addition to sorting bacterial display libraries, the commercial autoMCS instrument used here has also been used in part for the initial steps of discovery of human single-chain antibody fragments displayed on the surface of yeast, followed by further isolation using Fluorescent Activated Cell Sorting (FACS) 40. The primary advantages to semi-automation are decreased sorting time and effort, and more robust and reproducible elimination of unbound cells from the magnetic beads during wash steps, leading to reduced false positives, fewer required sorting rounds, and less complicated down-stream analysis 9,14. In the case of the commercial autoMCS instrument, there are multiple pre-loaded programs which may be optimal for different applications, such as negative pre-sorting (depletion), prioritizing purity, minimizing final sample volume, and increasing or decreasing sensitivity for isolation of rare cells 41.
The focus of this work is to demonstrate the methodology for biopanning a bacterial display library using the commercially available autoMCS instrument, and to expound the thought process applied in analyzing and minimizing the resulting pool of candidates in order to facilitate extension to other applications. The display library used here (see table of materials) 12,13 contains approximately 109- 1011 unique 15-mer peptides displayed via a modified version of the bacterial outer membrane protein OmpX in an unconstrained nature at the cell surface, which is expected to be representative of the free peptide in solution if produced synthetically. This protein scaffold additionally contains a positive control P2X peptide at the C-terminus for monitoring expression via binding to YPet Mona. However, a purchased or novel library with suitable diversity and other characteristics necessary for the specific application desired should work similarly with this biopanning procedure, though some minor changes may be required. A schematic of this biopanning protocol is illustrated in Figure 1. Additionally, the protocol could be adapted to other automated magnetic sorting platforms and other sorting strategies. Manual magnetic sorting with a benchtop magnet has been successfully demonstrated, albeit with more complicated and time consuming downstream analysis required due to increased false positives 14, and nonetheless provides an alternative option to the autoMCS steps if no automated magnetic cell sorting device is available.

<table><tbody><tr><td>autoMACS Pro Separator</td><td>Miltenyi Biotec</td><td>130-092-545</td><td>Automated cell separator for use with magnetic beads</td></tr><tr><td>Luria Broth, Miller (LB)</td><td>Fisher Scientific</td><td>BP1426-500</td><td>Growth medium</td></tr><tr><td>Chloramphenicol</td><td>Fisher BioReagents</td><td>BP904-100</td><td>Antibiotic</td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>BP2641-500</td><td>Thickening/geling agent</td></tr><tr><td>eCPX 3.0 Bacterial Display Peptide Library</td><td>Cytomx Therapeutics</td><td>N/A; http://cytomx.com/contact/</td><td>On-cell peptide display library. Provided by collaborators at USCB.</td></tr><tr><td>L-arabinose</td><td>Sigma</td><td>A3256-500G</td><td>Sugar, for induction of protein expression</td></tr><tr><td>Phosphate Buffered Saline pH 7.4 (PBS)</td><td>Amresco</td><td>0780-10L</td><td>Buffer</td></tr><tr><td>EZ-Link Sulfo-NHS-LC-Biotin, No-Weigh</td><td>Thermo Scientific</td><td>PI21327</td><td>Adds biotin molecule to primary amines</td></tr><tr><td>Pierce Biotin Quantitation Kit</td><td>Thermo Scientific</td><td>PI28005</td><td>Ensures biotin molecule is covalently bound to protein</td></tr><tr><td>Dynabeads MyOne Strepavidin T1 Beads</td><td>Invitrogen</td><td>65601</td><td>Magnetic beads for capture of biotin-congugated proteins</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A9647-100G</td><td>Protein, used as blocking agent for non-specific binding</td></tr><tr><td>D-glucose</td><td>Sigma</td><td>G8270-1KG</td><td>Sugar, for growth and impeding inducible protein expression</td></tr><tr><td>Ypet Mona</td><td>UCSB and ARL</td><td>N/A</td><td>Positive control for monitoring expression of eCPX 3.0. Produced by authors and collaborators; see references 12 and 13.</td></tr><tr><td>Dylight 488 NHS Ester</td><td>Thermo Scientific</td><td>46403</td><td>For conjugating protein target to fluorophore</td></tr><tr><td>Streptavidin, R-Phycoerythrin conjugate (SAPE)</td><td>Thermo Scientific</td><td>S866</td><td>Negative control for monitoring non-specific binding to streptavidin</td></tr><tr><td>BD FACSFlow</td><td>Becton, Dickinson, and Company</td><td>342003</td><td>Buffer for running FACS instrument</td></tr><tr><td>autoMACS Columns</td><td>Miltenyi Biotech</td><td>130-021-101</td><td>For MACS Separation</td></tr><tr><td>autoMACS Running Buffer &amp;ndash; MACS Separation Buffer</td><td>Miltenyi Biotech</td><td>130-091-221</td><td>For autoMACS instrument maintenance. Hazard: contains 0.09% azide.</td></tr><tr><td>autoMACS Pro Washing Solution</td><td>Miltenyi Biotech</td><td>130-092-987</td><td>For autoMACS instrument maintenance</td></tr><tr><td>70% Ethanol</td><td>VWR</td><td>E505-4L</td><td>Decontamination of autoMACS</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G6279-1L</td><td>For bacterial freezer stocks</td></tr><tr><td>Chill 15 rack</td><td>Miltenyi Biotec</td><td>130-092-952</td><td>For MACS Separation</td></tr><tr><td>BD FACSCanto II</td><td>Becton, Dickinson, and Company</td><td>744810; http://www.bdbiosciences.com/us/instruments/research/cell-analyzers/bd-facscanto-ii/m/744810/overview</td><td>For assessment of peptide binding to target</td></tr><tr><td>BD FACSDiva Software</td><td>Becton, Dickinson, and Company</td><td>659523</td><td>For assessment of peptide binding to target</td></tr><tr><td>Dynabeads MPC-S magnetic particle concentrator</td><td>Thermo Scientific</td><td>A13346</td><td>Bench top cell separator for use with magnetic beads</td></tr><tr><td>Colony Sequencing</td><td>Genewiz</td><td>N/A; https://www.genewiz.com/</td><td>DNA and Colony Sequencing Services</td></tr><tr><td>Excel</td><td>Microsoft</td><td>323021400; https://www.microsoftstore.com/store/msusa/en_US/pdp/Excel-2016/productID.323021400</td><td>For use with sequence analysis macro</td></tr><tr><td>Anthrax Protective Antigen (PA)</td><td>List Biological Laboratories, Inc.</td><td>171</td><td>Target protein used as example for representative results.</td></tr><tr><td>Abrax</td><td>ECBC and ARL</td><td>N/A</td><td>Target protein used as example for representative results. Produced by authors and collaborators; see references 1 and 15.</td></tr><tr><td>Rivax</td><td>ECBC and ARL</td><td>N/A</td><td>Target protein used as example for representative results. Produced by authors and collaborators; see references 1 and 15.</td></tr></tbody></table>

semi-automated biopanning of bacterial display libraries for peptide affinity reagent discovery and analysis of resulting isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Watch this Scientific Journal Video about Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates at JoVE.com

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

semi-automated-biopanning-bacterial-display-libraries-for-peptide

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.4K Views.  US Army Research Laboratory. Biopanning bacteriële display bibliotheken is een bewezen techniek voor ontdekking van peptide affiniteit reagentia, een robuust alternatief voor antilichamen. De semi-automatische sorteermethode hierin heeft gestroomlijnd biopanning als u wilt verkleinen van het optreden van valse positieven. We illustreren hier het denkproces en de technieken toegepast in de beoordeling van kandidaten en het minimaliseren van de downstream-analyse.

Video: Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Immunology and Infection

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Bioengineering

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic biotinylation by the E. coli biotin-protein ligase BirA is highly specific and allows for the biotinylation of target proteins in their native environment; however, the current usage of BirA mediated biotinylation requires the presence of a synthetic acceptor peptide (AP) in the target protein. Therefore, its application is limited to proteins that have been engineered to contain the AP. The purpose of the present protocol is to use the bacterial display of a peptide derived from an unmodified target protein to select for BirA variants that biotinylates the peptide. The system is based on a single plasmid that allows for the co-expression of BirA variants along with a scaffold for the peptide display on the bacterial surface. The protocol describes a detailed procedure for the incorporation of the target peptide into the display scaffold, creation of the BirA library, selection of active BirA variants and initial characterization of the isolated BirA variants. The method provides a highly effective selection system for the isolation of novel BirA variants that can be used for the further directed evolution of biotin-protein ligases that biotinylate a native protein in complex solutions.

Here we present a method to select for novel variants of the E. coli biotin-protein ligase BirA that biotinylates a specific target peptide. The protocol describes the construction of a plasmid for the bacterial display of the target peptide, generation of a BirA library, selection and characterization of BirA variants.

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Biology

A Peptide Microarray Technology for Specificity Profiling of Antibodies

Source: Cornett, E. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55912">Analysis of Histone Antibody Specificity with Peptide Microarrays.</a> J. Vis. Exp. (2017).
This video demonstrates a technique that applies peptide microarray technology to profile the specificity of antibodies recognizing post-translational modifications (PTMs) on histone peptides. A microarray slide containing immobilized fluorophore-conjugated histone peptides with known PTM combinations is treated with target PTM-specific antibodies. This process helps identify the antibody&#39;s specificity for the target and its potential cross-reactivity with non-target PTMs.

1. Designing the Array Slide and Source Plate Layout

	Under the &#39;Plan a slide layout&#39; heading on the ArrayNinja interface, click on the link that ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Quartz-Crystal Microbalance Biosensor-Based Biopanning to Study Protein-Drug Interactions

Source: Takakusagi, Y. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61873">Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions.</a> J. Vis. Exp. (2020)
This video demonstrates a method to study protein-drug interactions using QCM biosensor-based biopanning of the T7 phage-displayed peptide library. The biopanning of phages displaying a specific peptide on the QCM biosensor allows specific isolation of high-affinity peptides, followed by amino acid sequencing, which helps to identify specific interaction sites on the target protein.

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

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