We thank Tomer Zohar for work on scFv screening and characterization assays. A portion of this work was supported by award number F32CA150622 from the National Cancer Institute (to AJK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

<ol>
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The authors declare that they have no competing financial interests.

Engineering antibodies for cytoplasmic activity is a difficult task due to the reducing milieu of the cytoplasm, which impedes the formation of stabilizing disulfide bonds6,7. This causes most antibodies to be cytoplasmically inactive unless they are engineered for stability and solubility in the cytoplasm, in addition to being engineered for binding affinity. The existing methods of phage display, bacterial surface display, and yeast surface display methods all use the secretory pathway14-16 for the display of engineered antibodies, but these methods have no means to engineer intracellular folding. Antibodies engineered using inner-membrane display have improved cytoplasmic stability and solubility because the folding quality control of the Tat pathway prevents translocation of antibodies that are poorly folded and unstable in the cytoplasm. This method simplifies the iterative process of engineering intracellular antibodies for affinity and solubility, as the two properties are engineered in one step. Although this method was designed for engineering antibodies with solubility in the reducing intracellular environment, it could also be applied to engineering antibodies to function in non-reducing conditions, since the proteins engineered using this method maintain their folding in the oxidizing environment of the periplasm.
Although this technique simplifies the process of engineering antibodies with high affinity and high cytoplasmic solubility, several limitations are important to consider when using this protocol. When analyzing the secondary screen ELISA signals to identify promising scFv variants, the threshold for discerning between potentially interesting variants and those that may not exhibit adequate antigen-binding is not likely to be apparent until after several clones have been further characterized. It is important to look for improved binding over the parent antibody; however, an abnormally high signal could be indicative of avidity29 or aggregation effects30, a challenge which is not unique to the inner-membrane display screening approach. A key limitation to remember when using this protocol is the inability to recover spheroplasts after panning, as they are non-viable (unpublished data). This necessitates the DNA amplification and transformation steps to recover the antibody-encoding plasmids.
Several critical steps of the protocol enable the simultaneous engineering of folding and binding of antibodies. For screening to be successful, the scFv library being screened must be expressed as a fusion to the ssTorA signal peptide. Without this sequence, antibodies will not be directed to the Tat pathway and thus will not be translocated to the periplasm19. Additionally, it is imperative that a C-terminal epitope tag is fused to the antibodies to allow detection of the displayed antibodies in the binding assays. Clearly, the E. coli strain used to express the scFvs must also have the necessary Tat pathway machinery, but this is true of the commonly used E. coli strains.
Modifications to this protocol are possible to improve its potential to isolate antibodies with the desired characteristics. A subtractive panning step may be completed prior to panning against the target antigen to deplete the scFv library of non-desired constituents. The library spheroplasts can be incubated with magnetic beads coated with BCCP alone or coated with a non-desired protein, and the spheroplasts that bind to those beads can be discarded before screening the remaining unbound spheroplasts for binding to the desired target. As mentioned in the Representative Results, a method to improve the affinity of an isolated scFv is to include a soluble competitor in the panning reaction to compete with the scFvs displayed on the spheroplasts. Because the soluble competitor is a purified protein, no DNA is amplified from it, so only sequences of the scFvs displayed on the spheroplasts will be recovered in the PCR reaction. Additionally, this method could be extended to engineering other types of antibodies or to non-antibody binding proteins.
E. coli inner-membrane display is a powerful platform for engineering antibodies with high affinity and high levels of intracellular solubility. This method is particularly suited for efficient engineering of antibodies designed to function in the intracellular environment. These intracellular antibodies are already being explored as potential therapeutics in a number of fields, including neurodegenerative diseases, cancer, and viral infections31. This technique will enable more widespread use of intracellular antibodies as tools for research and medicine in these fields and any other field where studying a protein target in situ is desired.

Antibodies capable of folding and functioning in the intracellular environment are promising tools for both research and therapeutic applications. They have the ability to modulate protein activity by binding to a target protein inside cells to prevent protein-protein interactions, disrupt protein-nucleic acid interactions, or prevent substrate access to enzymes1-5.
Although antibodies have much potential for intracellular applications, engineering them for proper folding and solubility in the intracellular environment while maintaining the ability to bind to a target antigen is challenging. The reducing cytoplasmic environment prevents the formation of the disulfide bonds normally required for the stable folding of full-length antibodies and antibody fragments, including single-chain variable fragment (scFv) antibodies6,7. A number of directed evolution approaches have been employed to engineer antibodies with high affinities for target antigens8-10. These approaches commonly use phage display, yeast surface display, or bacterial surface display to screen large libraries of antibodies11-13. These methods are powerful and effective for identifying antibodies that bind to targets, yet they depend on the secretory pathway to transport proteins that will be displayed14-16. The secretory pathway translocates unfolded proteins from the reducing cytoplasm into the endoplasmic reticulum lumen in yeast or into the periplasm in bacteria. The proteins then fold under oxidizing conditions and are displayed on the cell surface or packaged into phage particles to screen for binding affinity17,18. As a result, antibodies isolated using these techniques will not necessarily fold well in the cytoplasm, and intracellular solubility must often be engineered separately if the antibodies will be used in intracellular applications.
To improve the efficiency of engineering antibodies that are well folded in the cytoplasm, we previously reported the success of MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins), a method for screening an scFv antibody library using Escherichia coli inner-membrane display19. Bacterial inner-membrane display relies on the twin-arginine translocation (Tat) pathway for transporting displayed antibodies, in contrast to other common display methods that use the secretory pathway. The Tat pathway contains a quality control mechanism that only allows soluble, correctly folded proteins to be transported from the E. coli cytoplasm, across the inner membrane, and into the periplasm20,21. Overexpressed Tat substrates (i.e., proteins targeted to the Tat pathway with an N-terminal fusion to the Tat signal peptide ssTorA) that are well folded in the cytoplasm form a long-lived translocation intermediate with the N-terminus in the cytoplasm and the C-terminus in the periplasm19. This allows display of correctly folded Tat substrates, including antibody fragments, on the periplasmic face of the E. coli inner membrane. After removing the outer membrane by enzymatic digestion to generate spheroplasts, antibodies are exposed to the extracellular space (Figure 1). This allows Tat substrates displayed on the inner membrane to be screened for binding to a specific target. Importantly, harnessing the Tat pathway for cell-surface display ensures that only the antibodies in the library that are well folded in the cytoplasm will be interrogated for binding, allowing simultaneous engineering of binding affinity and intracellular folding. In this protocol, we describe how to display an scFv library on the E. coli inner membrane, pan the library against a target antigen, and perform a secondary screen to identify the most promising constituents of the library. While we focus the protocol on scFvs, the method could be applied to engineering any protein whose application requires binding and intracellular folding.
<img alt="Figure 1" src="/files/ftp_upload/54583/54583fig1.jpg" /> 
Figure 1. Tat inner-membrane display. In E. coli, scFv antibodies that are expressed as a fusion to the ssTorA signal sequence and correctly folded in the cytoplasm are transported across the inner membrane. A translocation intermediate forms, where the scFvs are anchored in the inner membrane with the N-terminus in the cytoplasm and the C-terminus in the periplasm. The E. coli outer membrane is enzymatically digested to form spheroplasts, thereby exposing the anchored antibodies to the extracellular space and making them available for detection by using an antibody that binds to the C-terminally fused epitope tag on the displayed antibody. <a href="http://www.jove.com/files/ftp_upload/54583/54583fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>scFv library</td><td>Varies</td><td></td><td>A suitable scFv library should be obtained from a commercial or academic source.</td></tr><tr><td>MC4100 &lt;em&gt;E. coli&lt;/em&gt; cells</td><td>Coli Genetic Stock Center</td><td>6152</td><td>Cells need to be chemically competent or electrocompetent, depending on the selected transformation method.</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-4</td><td></td></tr><tr><td>Difco dehydrated culture media LB Broth, Miller (Luria-Bertani)</td><td>BD</td><td>244610</td><td></td></tr><tr><td>Chloramphenicol (Cm)</td><td>Fisher Scientific</td><td>BP904-100</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Fisher Scientific</td><td>BP366-500</td><td></td></tr><tr><td>Sodium phosphate, dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>Potassium phosphate, monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>BP362-500</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher Scientific</td><td>BP9706-100</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>BP220-1</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP1521</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA), 0.5 M</td><td>Fisher Scientific</td><td>BP2482-500</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>BP214-500</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma Aldrich</td><td>L3790-10X1ML</td><td></td></tr><tr><td>Vortex mixer</td><td>VWR</td><td>97043-564</td><td></td></tr><tr><td>Bicine</td><td>Fisher Scientific</td><td>BP2646100</td><td></td></tr><tr><td>D-Biotin</td><td>Fisher Scientific</td><td>BP232-1</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside</td><td>Fisher Scientific</td><td>BP1755-1</td><td></td></tr><tr><td>BugBuster Master Mix (cell lysis detergent)</td><td>EMD Millipore</td><td>71456</td><td></td></tr><tr><td>Vivaspin 2 MWCO, 3,000&amp;nbsp;daltons</td><td>GE Healthcare Sciences</td><td>28932240</td><td></td></tr><tr><td>Target antigen</td><td>Varies</td><td>N/A</td><td>Purified target antigen may be purchased or produced/purified.</td></tr><tr><td>Dynabeads MyOne Streptavidin T1</td><td>Invitrogen</td><td>65601</td><td></td></tr><tr><td>Dynamag-2 magnet</td><td>Invitrogen</td><td>12321D</td><td></td></tr><tr><td>Tube rotator</td><td>VWR</td><td>13916-822</td><td></td></tr><tr><td>PCR primers</td><td>IDT</td><td>N/A</td><td>Primer sequences are as described in the protocol.</td></tr><tr><td>10x T4 DNA ligase reaction buffer</td><td>New England BioLabs</td><td>B0202S</td><td></td></tr><tr><td>T4 Polynucelotide kinase (PNK)</td><td>New England BioLabs</td><td>M0201S</td><td>Make sure the T4 ligase buffer used in the primer phosphorylation reaction contains 1 mM ATP.&amp;nbsp;</td></tr><tr><td>5x Phusion HF buffer pack</td><td>New England BioLabs</td><td>B0518S</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) solution mix, 10 mM each dNTP</td><td>New England BioLabs</td><td>N0447L</td><td></td></tr><tr><td>Phusion DNA polymerase</td><td>New England BioLabs</td><td>M0530S</td><td>Other high-fidelity polymerases may be used as an alternative, but the annealing temperature in &lt;strong&gt;Table 3&lt;/strong&gt; must be adjusted.</td></tr><tr><td>C1000 Touch thermal cycler with dual 48/48 fast reaction module</td><td>Bio-Rad</td><td>185-1148</td><td></td></tr><tr><td>Agarose</td><td>Promega</td><td>V3121</td><td></td></tr><tr><td>SYBR Safe DNA gel stain</td><td>Invitrogen</td><td>S33102</td><td></td></tr><tr><td>Wizard SV gel and PCR clean-up system</td><td>Promega</td><td>A9281</td><td></td></tr><tr><td>T4 DNA ligase</td><td>New England BioLabs</td><td>M0202S</td><td></td></tr><tr><td>Microdialysis membrane filter</td><td>EMD Millipore</td><td>VSWP04700</td><td></td></tr><tr><td>Agar</td><td>BD</td><td>214030</td><td></td></tr><tr><td>96-well polystyrene round-bottom cell culture plates</td><td>VWR</td><td>10062-902</td><td></td></tr><tr><td>Costar general polystyrene assay plate lids</td><td>Corning</td><td>3931</td><td></td></tr><tr><td>Microtitre plate shaker</td><td>VWR</td><td>12620-926</td><td></td></tr><tr><td>Costar 96 well EIA/RIA Easy Wash clear flat bottom polystyrene high bind microplate</td><td>Corning</td><td>3369</td><td></td></tr><tr><td>Bel-blotter polycarbonate 96-well replicating tool</td><td>Bel-Art Products</td><td>378760002</td><td></td></tr><tr><td>Instant nonfat dry milk</td><td>Quality Biological</td><td>A614-1000</td><td></td></tr><tr><td>Tween 20 (polysorbate 20)</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>PopCulture reagent (concentrated cell lysis detergent)</td><td>EMD Millipore</td><td>71092-3</td><td></td></tr><tr><td>Monoclonal ANTI-FLAG M2-Peroxidase(HRP) antibody produced in mouse</td><td>Sigma Aldrich</td><td>A8592</td><td></td></tr><tr><td>SigmaFast OPD</td><td>Sigma Aldrich</td><td>P9187-50SET</td><td></td></tr><tr><td>Sulfuric acid (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;), 10&amp;nbsp;N solution</td><td>Fisher Scientific</td><td>SA200-1</td><td></td></tr><tr><td>Reynolds Wrap aluminum foil</td><td>VWR</td><td>89079-075</td><td></td></tr><tr><td>BioTek Epoch microplate spectrophotometer</td><td>Fisher Scientific</td><td>11120570</td><td></td></tr></tbody></table>

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.

The intracellular protein folding quality control mechanism of the Tat pathway in E. coli limits transport across the inner cell membrane to proteins that are well folded in the reducing cytoplasmic environment. By overexpressing a fusion of an scFv to the ssTorA signal sequence (the signal sequence from the TorA protein, which is naturally transported by the Tat pathway20), translocation is stalled, resulting in display of scFvs on the inner membrane19. After enzymatic disruption of the outer membrane, the displayed antibodies are made available for screening for antigen-binding activity. The ability to take advantage of the Tat pathway for scFv display was shown by Karlsson et al.19 (Figure 6). The scFv antibodies scFv13 and scFv13.R4 were fused to either the native ssTorA sequence or a modified ssTorA that lacks the arginine-arginine residue pair recognized by the Tat pathway. scFv13.R4 was engineered by Martineau et al. from scFv13 through four rounds of directed evolution and is known to fold well in the cytoplasm9. This scFv was displayed on the inner membrane, but only when expressed as a fusion to the native ssTorA signal sequence (Figure 6). Contrarily, scFv13 is not well folded cytoplasmically9, so it is not displayed well on the inner membrane, regardless of the signal sequence to which it is fused. Additionally, if the scFvs were expressed in cells that lacked the TatC protein, a vital component of the Tat machinery20,28, display was not observed, showing the important link between inner-membrane display and the Tat pathway. These results demonstrate that only proteins that contain the Tat signal peptide and that are correctly folded in the cytoplasm are displayed on the inner membrane, allowing transport through the Tat pathway to function as a screen for intracellular folding.
<img alt="Figure 6" src="/files/ftp_upload/54583/54583fig6.jpg" /> 
Figure 6. Detection of displayed scFvs on the inner membrane. Flow cytometry analysis was performed to detect the display of poorly folded scFv13 and well-folded scFv13.R4 on the inner membrane. scFvs were fused to native ssTorA or ssTorA(KK), where the Arg-Arg pair in the ssTorA sequence was modified to Lys-Lys. The C-terminal FLAG epitope tags on the scFvs were detected with a fluorescein isothiocyanate (FITC)-conjugated anti-FLAG antibody. Cells without the TatC protein (&#916;tatC) and ssTorA-scFv13 without the FLAG tag were tested as controls. M indicates the median fluorescence value. Reprinted from reference 19 with permission. <a href="https://www.jove.com/files/ftp_upload/54583/54583fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Inner-membrane display can successfully isolate scFv antibodies with high levels of affinity for a target protein and high levels of cytoplasmic solubility. Additionally, subsequent rounds of directed evolution using inner-membrane display improve antibody characteristics19. To demonstrate this, an error-prone PCR library based on scFv13, which has a low level of binding affinity for &#946;-galactosidase, was panned against the target antigen &#946;-galactosidase using the display and panning method described in the protocol. scFv 1-4 was isolated after one round of mutagenesis and panning, and exhibited higher binding affinity to &#946;-galactosidase than scFv13 (Figure 7A) and a higher level of cytoplasmic solubility (Figure 7B).
A new library, based on scFv 1-4, was made using error-prone PCR, and panning of this second-generation library against &#946;-galactosidase was done using a modification of the described protocol. The panning against &#946;-galactosidase for the second round of evolution was done in the presence of purified, soluble scFv 14 as a competitor to improve the likelihood of isolating clones with higher affinity than scFv 1-4. After this second round of mutagenesis and panning, scFv 2-1 and scFv 2-3 were isolated using the ELISA-based secondary screening. These scFvs not only exhibited higher binding affinity for &#946;-galactosidase than scFv13, but also exhibited better binding than the first-round clone scFv 1-4. scFv 2-1 exhibited &#946;-galactosidase binding comparable to that of scFv13.R4 (Figure 7A). scFv 2-3 also shows a further increase in cytoplasmic solubility compared to scFv 14, highlighting the simultaneous engineering of solubility and antigen-binding. Since affinity and soluble expression of the scFvs are screened for simultaneously, it is possible that a selected scFv has moderate solubility but high binding or vice versa. For example, scFv 2-1 has lower soluble expression than scFv 2-3, but it exhibits higher binding affinity to &#946;-galactosidase.
<img alt="Figure 7" src="/files/ftp_upload/54583/54583fig7.jpg" /> 
Figure 7. Target-binding and cytoplasmic expression of scFv variants isolated using inner-membrane display. (A) scFvs were expressed in the cytoplasm of E. coli cells (e.g., without the ssTorA signal sequence) with a hexahistidine (6&#215;-His) tag and purified using nickel-nitrilotriacetic acid spin-columns. The binding of the purified scFvs to &#946;-galactosidase was measured with an ELISA. Purified scFvs were loaded onto &#946;-galactosidase-coated ELISA plates, and the bound scFvs were detected with an anti-6&#215;-His antibody. The data are an average of six replicates, and the error bar shows standard error of the mean. (B) The soluble and insoluble fractions of the cell lysates from cells expressing the scFvs cytoplasmically were analyzed by a Western blot probed with an anti-6&#215;-His antibody. Total protein concentration was used to normalize the loading of the samples. Reprinted (A) and adapted (B) from reference 19 with permission. <a href="https://www.jove.com/files/ftp_upload/54583/54583fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments at JoVE.com

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

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 ...

bacterial-inner-membrane-display-for-screening-library-antibody

Research

JoVE Journal

Biochemistry

11.4K Views.  University of Maryland. 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.

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

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.

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. ...

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

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

Author Spotlight: Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Developmental Biology

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

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 ...

Optimizing Protein Functions Through Yeast Surface Display Techniques

Protein Engineering by Yeast Surface Display

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and versatile tools in the protein engineering field is yeast surface display, where a pool of randomized proteins is expressed on the surface of yeast. The linkage of phenotype (e.g., binding of the yeast-displayed protein to the antigen of interest) and genotype (the plasmid encoding for the protein variant) enables selection of this library for desired properties and subsequent sequencing of enriched variants. By combining magnetic bead selection with flow cytometric sorting, protein variants with enhanced binding to a target antigen can be selected and enriched. Notably, in addition to affinity maturation, binding to a target can also be achieved without any initial binding affinity. Here, we provide a step-by-step protocol that covers all essential parts of a yeast surface display selection campaign and gives examples of typical yeast surface display results. We demonstrate that yeast surface display is a broadly applicable and robust method that can be established in any molecular biology laboratory with access to flow cytometry.

This protocol describes the essential steps for conducting yeast surface display selection campaigns to enrich protein variants binding to an antigen of interest.

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and ...

Bioengineering

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...