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 &#8211; 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

Research

JoVE Journal

Biochemistry

11.3K Views.  US Army Research Laboratory. 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.

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 ...

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

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 ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) hydration, mucus viscosity and activity of antimicrobial peptides, key parameters involved in innate defense of the lungs. This is of primary relevance in the field of chronic respiratory diseases such as cystic fibrosis (CF) where these parameters are dysregulated. While different groups have studied ASL pH both in vivo and in vitro, their methods report a relatively wide range of ASL pH values and even contradictory findings regarding any pH differences between non-CF and CF cells. Furthermore, their protocols do not always provide enough details in order to ensure reproducibility, most are low throughput and require expensive equipment or specialized knowledge to implement, making them difficult to establish in most labs. Here we describe a semi-automated fluorescent plate reader assay that enables the real-time measurement of ASL pH under thin film conditions that more closely resemble the in vivo situation. This technique allows for stable measurements for many hours from multiple airway cultures simultaneously and, importantly, dynamic changes in ASL pH in response to agonists and inhibitors can be monitored. To achieve this, the ASL of fully differentiated primary human airway epithelial cells (hAECs) are stained overnight with a pH-sensitive dye in order to allow for the reabsorption of the excess fluid to ensure thin film conditions. After fluorescence is monitored in the presence or absence of agonists, pH calibration is performed in situ to correct for volume and dye concentration. The method described provides the required controls to make stable and reproducible ASL pH measurements, which ultimately could be used as a drug discovery platform for personalized medicine, as well as adapted to other epithelial tissues and experimental conditions, such as inflammatory and/or host-pathogen models.

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins with high specificity but low affinity. Through phage display, ubiquitin variants (UbVs) can be engineered such that they exhibit improved affinity over wildtype ubiquitin and maintain binding specificity to target proteins. Phage display utilizes a phagemid library, whereby the pIII coat protein of a filamentous M13 bacteriophage (chosen because it is displayed externally on the phage surface) is fused with UbVs. Specific residues of human wildtype ubiquitin are soft and randomized (i.e., there is a bias towards to native wildtype sequence) to generate UbVs so that deleterious changes in protein conformation are avoided while introducing the diversity necessary for promoting novel interactions with the target protein. During the phage display process, these UbVs are expressed and displayed on phage coat proteins and panned against a protein of interest. UbVs that exhibit favorable binding interactions with the target protein are retained, whereas poor binders are washed away and removed from the library pool. The retained UbVs, which are attached to the phage particle containing the UbV's corresponding phagemid, are eluted, amplified, and concentrated so that they can be panned against the same target protein in another round of phage display. Typically, up to five rounds of phage display are performed, during which a strong selection pressure is imposed against UbVs that bind weakly and/or promiscuously so that those with higher affinities are concentrated and enriched. Ultimately, UbVs that demonstrate higher specificity and/or affinity for the target protein than their wildtype counterparts are isolated and can be characterized through further experiments.

Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins ...

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...