Joshua M. Kogot

Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge

Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3×1010 members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS.

Increased affinity and solubility of peptides used for direct peptide ELISA on polystyrene surfaces through fusion with a polystyrene-binding peptide tag

Peptide reagents can serve as alternatives or replacements to antibodies in sensing or diagnostic applications. The passive adsorption of peptides onto polystyrene surfaces can limit the target binding capability, especially for short, positively charged, or hydrophobic sequences. In this report, we show that fusing a peptide with a previously characterized 12-amino acid polystyrene binding sequence (PS-tag) improves overall peptide solubility and enzyme-linked immunosorbent assay (ELISA) results using the peptide as a capture agent. Specific improvements for protective antigen (PA; Bacillus anthracis) protein binding peptides selected from bacterial surface display were compared with native or biotinylated peptides. The PS-tag was added to either peptide terminus, using a (Gly)(4) spacer, and comparable binding affinities were obtained. Fusion with the PS-tag did not have any negative impact on peptide secondary structure as measured by circular dichroism. The addition of the PS-tag provides a convenient method to utilize peptide reagents from peptide display libraries as capture agents in an ELISA format without the need for a biotin tag or concerns about passive adsorption of critical residues for target capture.

Bacterial Display Peptides for Use in Biosensing Applications

Peptide affinity reagent technology including bacterial display encapsulates an in vitro analog of classical Darwinian evolution through a physical linkage between polypeptide sequence and the encoding of genetic information. Similar to the inate human imune system a unique binder is isolated, however, in this directed evolution method a large library displaying billions of diverse peptide sequences is created and the sythetic binder to the target of interest is determined. Subsequently the synthetic reagent is mass-produced for use in the specific biosensing application (Park & Cochran, 2010; Stratis-Cullum & Sumner 2008). Currently, a number of systems, including messenger ribonucleic acid (mRNA) and ribosome display (Wilson, Keefe, & Szostak, 2001 ), eukaryotic virus display (Bupp and Roth, 2002; Muller, 2003), and bacterial and yeast surface display (Georgiou et al., 1997, Boder & Wittrup, 1997), are used to rapidly generate affinity reagents that can be used for diagnostics, proteomics, and therapeutic applications (Kodadek, 2001; Nixon, 2002). Bacterial cell surface display is advantageous because the use of bacterial cells simplifies the polypeptide selection method and enables fast screening of potential recognition elements typically using fluorescence activated cell-sorting (FACS) and/or magnetic activated cellsorting (MACS). Polypeptide affinity reagents can offer more stable alternatives to antibody technology, enabling more rugged application in the field (e.g., enhanced thermal stability). A key advantage of bacterial display technology over traditional antibody creation as well as other peptide display alternatives is that it offers an strategy for generating tailor-made affinity ligands in a very short time period of several days (Georgiou et al., 1997; Bessette, Rice and Daugherty 2004), see Figure 1. The development of a bacterial display system suitable for robust reagent discovery has proven challenging (Lee, Choi & Xu, 2003). There are three main challenges in creating a high speed affinity ligand isolation technology against unknown/un-catalogued pathogens: (1) creation of a large (high diversity) and robust cell based library, (2) creation of an ultrahigh throughput, disposable screening system, and (3) gaining a fundamental understanding of the factors which influence binder performance along with this understanding the development of methodologies to enable universal isolation and optimization of ligand binder performance.

Screening and characterization of anti-SEB peptides using a bacterial display library and microfluidic magnetic sorting

Bacterial peptide display libraries enable the rapid and efficient selection of peptides that have high affinity and selectivity toward their targets. Using a 15‐mer random library on the outer surface of Escherichia coli (E.coli), high‐affinity peptides were selected against a staphylococcal enterotoxin B (SEB) protein after four rounds of biopanning. On‐cell screening analysis of affinity and specificity were measured by flow cytometry and directly compared to the synthetic peptide, off‐cell, using peptide‐ELISA. DNA sequencing of the positive clones after four rounds of microfluidic magnetic sorting (MMS) revealed a common consensus sequence of (S/T)CH(Y/F)W for the SEB‐binding peptides R338, R418, and R445. The consensus sequence in these bacterial display peptides has similar amino acid characteristics with SEB peptide sequences isolated from phage display. The Kd measured by peptide‐ELISA off‐cell was 2.4 nM for R418 and 3.0 nM for R445. The bacterial peptide display methodology using the semiautomated MMS resulted in the discovery of selective peptides with affinity for a food safety and defense threat. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Journal of Molecular Recognition published by John Wiley & Sons, Ltd.

Development of Bacterial Display Peptides for use in Biosensing Applications

Recent advances in synthetic library engineering continue to show promise for the rapid production of reagent technology in response to biological threats. A synthetic library of peptide mutants built off a bacterial host offers a convenient means to link the peptide sequence, (i.e., identity of individual library members) with the desired molecular recognition traits, but also allows for a relatively simple protocol, amenable to automation. An improved understanding of the mechanisms of recognition and control of synthetic reagent isolation and evolution remain critical to success. In this paper, we describe our approach to development of peptide affinity reagents based on peptide bacterial display technology with improved control of binding interactions for stringent evolution of reagent candidates, and tailored performance capabilities. There are four key elements to the peptide affinity reagent program including: (1) the diverse bacterial library technology, (2) advanced reagent screening amenable to laboratory automation and control, (3) iterative characterization and feedback on both affinity and specificity of the molecular interactions, and (3) integrated multiscale computational prescreening of candidate peptide ligands including in silico prediction of improved binding performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be presented. Recent highlights of on cell vs. off-cell affinity behavior and correlation of the results with advanced docking simulations on the protein-peptide system(s) are included. The potential of this technology and approach to enable rapid development of a new affinity reagent with unprecedented speed (less than one week) would allow for rapid response to new and constantly emerging threats.

Isolation and characterization of anti-SEB peptides using magnetic sorting and bacterial peptide display library technology

Peptide display libraries offer an alternative method to existing antibody development methods enabling rapid isolation of highly stable reagents for detection of new and emerging biological threats. Bacterial display libraries are used to isolate new peptide reagents within 1 week, which is simpler and timelier than using competing display library technology based on phage or yeast. Using magnetic sorting methods, we have isolated peptide reagents with high affinity and specificity to staphylococcal enterotoxin B (SEB), a suspected food pathogen. Flow cytometry methods were used for on-cell characterization and the binding affinity (Kd) of this new peptide reagent was determined to be 56 nm with minimal cross-reactivity to other proteins. These results demonstrated that magnetic sorting for new reagents using bacterial display libraries is a rapid and effective method and has the potential for current and new and emerging food pathogen targets.

Empirical Methods for Identifying Specific Peptide-protein Interactions for Smart Reagent Development

The current state of the art in the development of antibody alternatives is fraught with difficulties including mass production, robustness, and overall cost of production. The isolation of synthetic alternatives using peptide libraries offers great potential for recognition elements that are more stable and have improved binding affinity and target specificity. Although recent advances in rapid and automated discovery and synthetic library engineering continue to show promise for this emerging science, there remains a critical need for an improved fundamental understanding of the mechanisms of recognition. To better understand the fundamental mechanisms of binding, it is critical to be able to accurately assess binding between peptide reagents and protein targets. The development of empirical methods to analyze peptide-protein interactions is often overlooked, since it is often assumed that peptides can easily substitute for antibodies in antibody-derived immunoassays. The physico-chemical difference between peptides and antibodies represents a major challenge for developing peptides in standard immunoassays as capture or detection reagents. Analysis of peptide presents a unique challenge since the peptide has to be soluble, must be capable of target recognition, and capable of ELISA plate or SPR chip binding. Incorporating a plate-binding, hydrophilic peptide fusion (PS-tag) improves both the solubility and plate binding capability in a direct peptide ELISA format. Secondly, a solution based methods, affinity capillary electrophoresis (ACE) method is presented as a solution-based, affinity determination method that can be used for determining both the association constants and binding kinetics.