Advances in Synthetic Peptides Reagent Discovery

by Bryn L. Adams, Deborah A. Sarkes, Amethist S. Finch, and 

Dimitra N. Stratis-Cullum

ARL-RP-0451 July 2013

A reprint from In Proc. SPIE 8710 Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) 

Sensing XIV, edited by Augustus Way Fountain, Proc. of SPIE Vol. 8710, 87101B.

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ARL-RP-0451 July 2013

Advances in Synthetic Peptides Reagent Discovery

Bryn L. Adams, Deborah A. Sarkes, Amethist S. Finch, and 

Dimitra N. Stratis-Cullum

Sensors and Electron Devices Directorate, ARL

A reprint from In Proc. SPIE 8710 Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) 

Sensing XIV, edited by Augustus Way Fountain, Proc. of SPIE Vol. 8710, 87101B.

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A reprint from In Proc. SPIE 8710 Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XIV, 

edited by Augustus Way Fountain, Proc. of SPIE Vol. 8710, 87101B.

14. ABSTRACT

Bacterial display technology offers a number of advantages over competing display technologies (e.g, phage) for the rapid 

discovery and development of peptides with interaction targeted to materials ranging from biological hazards through 

inorganic metals. We have previously shown that discovery of synthetic peptide reagents utilizing bacterial display technology 

is relatively simple and rapid to make laboratory automation possible. This included extensive study of the protective antigen 

system of Bacillus anthracis, including development of discovery, characterization, and computational biology capabilities for 

in-silico optimization. Although the benefits towards CBD goals are evident, the impact is far-reaching due to our ability to 

understand and harness peptide interactions that are ultimately extendable to the hybrid biomaterials of the future. In this 

paper, we describe advances in peptide discovery including, new target systems (e.g. non-biological materials), advanced 

library development and clone analysis including integrated reporting.

15. SUBJECT TERMS

peptide, synthetic reagent, affinity reagent, bacterial display, biosensing, hybrid materials, advanced libraries

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Advances in synthetic peptides reagent discovery 

Bryn L. Adams, Deborah A. Sarkes, Amethist S. Finch, and Dimitra N. Stratis-Cullum*

U.S. Army Research Laboratory

ABSTRACT

Bacterial display technology offers a number of advantages over competing display technologies (e.g, phage) for the 

rapid discovery and development of peptides with interaction targeted to materials ranging from biological hazards 

through inorganic metals. We have previously shown that discovery of synthetic peptide reagents utilizing bacterial 

display technology is relatively simple and rapid to make laboratory automation possible. This included extensive study 

of the protective antigen system of Bacillus anthracis, including development of discovery, characterization, and 

computational biology capabilities for in-silico optimization. Although the benefits towards CBD goals are evident, the 

impact is far-reaching due to our ability to understand and harness peptide interactions that are ultimately extendable to 

the hybrid biomaterials of the future. In this paper, we describe advances in peptide discovery including, new target 

systems (e.g. non-biological materials), advanced library development and clone analysis including integrated reporting.

Keywords: peptide, synthetic reagent, affinity reagent, bacterial display, biosensing, hybrid materials, advanced libraries

1. INTRODUCTION

Persistent surveillance of a variety of threat agents at trace concentrations, whether chemical or biological in nature, in 

air, food or water supplies is of critical to ensure the highest degree of survivability across the spectrum of operations of 

our nation’s soldiers. One of the key challenges in hazard detection is that threat agents do not exist in an isolated 

environment, but are contained within a complex background of interfering molecules, which impedes rapid and 

accurate sensing. In order to overcome these challenges, there is a need to develop bioreceptors (i.e. affinity reagents) 

with specific recognition for the target of interest, that are robust and reliable. Many sensor technologies utilize 

antibodies, which have high affinity and specificity to a given target, but are very costly, difficult to mass-produce and 

typically have limited stability. Additionally, the current rate of antibody production (weeks to months) does not meet 

the rate the DOD requires for reagent development against new and emerging threats. Alternatively, synthetic affinity 

reagents have been developed from various synthetic recognition element technologies (e.g. bacterial display and phage 

display) in a matter of days or weeks [1]. 

Synthetic recognition elements (SRE) include various types of biological molecules, such as nucleic acids, peptides, and 

proteins, and have been extensively reviewed[2, 3]. Our work has primarily focused on the development and use of 

peptide-based SRE. Like the traditional antibody-antigen interactions, peptide SRE or peptide affinity reagents, 

selectively bind to a target with high affinity via a mechanism similar to antibodies. However, unlike antibodies, these 

reagents can be developed very rapidly and offer a level of stability not possible with standard antibodies. Specific 

affinity peptides are developed by screening a library of peptides for binders to a target of interest and once identified, 

those peptides can be mass produced through standard synthetic techniques. 

Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XIV, 

edited by Augustus Way Fountain, Proc. of SPIE Vol. 8710, 87101B · © 2013 

SPIE · CCC code: 0277-786X/13/$18 · doi: 10.1117/12.2017089

Proc. of SPIE Vol. 8710 87101B-1

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Several types of cell surface display systems have been developed, including phage [4], bacteria [5-7], yeast [8, 9], 

insect cell/baculovirus [10], as well as mammalian cell culture [11]. Although each system has its unique advantages and 

disadvantages, they are all used in conjunction with biopanning to identify affinity peptides to a specific target. 

Biopanning is an affinity selection technique in which a library of cell surface displayed peptides is incubated with the 

target and binding sequences are screened and selected for further propagation. By re-screening this selected 

subpopulation of binders under more stringent conditions, higher affinity binders can be enriched. A final set of target 

binders can be identified through DNA sequencing, as the peptide sequence is DNA encoded [12]. Compared to the 

other display technologies, bacterial display, specifically Escherichia coli-based display systems, have an extremely fast 

replication rate and can easily be genetically modified for tailored biodiscovery applications. Thus, bacterial display is 

ideal for combating newly emerging threats that have no readily available or specific antibody bioreceptor. 

E. coli has been engineered to develop affinity peptides by modifying natural cell components or systems. Brown [13]

developed an E. coli concatamer library displayed on the external domain of a lambda phage receptor and the adherence 

appendage, fimbriae, was modified in another E. coli to form a display scaffold by inserting a peptide library into the 

structural component, FimH [14]. To date, the most widely used E. coli surface display system is FliTrx. This E. coli

flagella-based display system has been used to develop a variety of peptide SRE’s to diverse targets [7] [15-18]. 

Although these systems have been successfully utilized to produce affinity peptide reagents, they are often criticized for 

their lack of peptide diversity as compared to other display technologies (e.g. phage display). Recently, a novel, 

engineered E. coli peptide display library has been developed with a greater estimated diversity (3x1010 discreet random 

peptides) [19, 20]. This library utilizes the engineered protein, eCPX, as a display scaffold. This is a circularly permuted 

form of the native E. coli outer membrane protein, OmpX. The eCPX protein is a membrane spanning beta barrel with a 

surface exposed C- and N-terminus. The C-terminus contains a P2X peptide, which binds the Mona-SH3 domain [21]

and the N-terminus contains a flexible random 15 amino acid peptide library [19, 20] (Fig. 1). This system has been

previously used to successfully screen for peptides SRE’s to streptavidin [19], protease activated proligands [22], proapoptotic and anti-apoptotic peptides [23, 24], vascular endothelial growth factor (VEGF) [21], breast tumor type–

specific peptide ligands [25], protective antigen (PA) protein of Bacillus antracis [1, 26-29], and staphylococcal 

enterotoxin B (SEB) [30].

Figure 1. Conceptual diagram of an unconstrained bacterial display technology possessing an engineered, 

randomized 15-mer amino acid binding region and a simultaneous tag to monitor expression enabling 

quantitative affinity reagent screening.

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A I E. coil eCPX peptide

display

4 Incubation with protein

target bound to magnetic

bead

t

Repeat screening wtth lower

target protein concentration

to increase affinity selection

t

z

Screen for cell binders

to target. wash away

unbound cells

Detach cells from magnetic

beads and propagate binders

1

Isolate cells and sequence DNA to

identify pegt.ides

ll

Rand Rari! 2 Rand 3

Biopanning Rounds

2. ADVANCES IN BACTERIA SURFACE DISPLAY BIOPANNING 

2.1 Traditional biopanning

Bacterial peptide display is most commonly used to isolate binders to proteins, and has been used for applications such 

as vaccine development [31], biocatalysts [32], and biosensors [33]. As such, many of the bacterial display libraries 

were develop and biopanning techniques optimized for use with a protein target. Although the exact steps may vary for 

each type of bacterial display system, the general biopanning process is very similar and is depicted in Fig. 2A. 

Biopanning consists of incubating the library with a protein of interest and selecting those cells that bound the target. 

Due to the small size and ease of “tagging” protein targets, a variety of commercial and custom technologies are 

available to aid in high throughput screening and selection of target binding cells. These steps are typically performed 

one of two ways: 1) the target protein is fluorescently labeled and fluorescence activated cell sorting (FACS) is used to 

extract those bound cells from the population or 2) the target protein is conjugated to magnetic beads, which retain the 

protein bound cells while non-binders are washed away. Both cell binder selection approaches have been shown 

successful in previous studies [20, 26]. A modified, self contained, automatic magnetic selection technique was develop 

and described by Kogot et al. [26]. A Micro-Magnetic Separation (MMS) platform was used to screen the eCPX library 

for peptide SRE’s to the PA protein to improve the reproducibility, reduce the cost, reduce cross contamination, and 

minimize exposure to the target protein. 

After selection of target binding cells, this target affinity subpopulation is then propagated to create a secondary cell 

population to further refine for higher target affinity binders. By repeating the screening and selection steps (rounds) 

utilizing sequentially lower concentrations of the target protein, the highest affinity peptides are selected. Typically, 3-5 

rounds of biopanning are sufficient for the completion of affinity peptide biodiscovery. The affinity maturation of the 

enriched surface displayed peptide population can be seen by measuring the number of cells binding the protein target in 

each round. For example, in the development of PA peptide SRE’s from the eCPX library, marked increases in the 

Figure 2. (A) Schematic diagram showing the steps involved in traditional biopanning of a display library 

including incubation with magnetic beads conjugated to the protein target, stringency wash steps to 

remove weakly bound library members, and enrichment of the bound population prior to identification of 

isolated colonies using DNA sequencing. (B) Shows a typical enrichment in the % of a library population 

that binds over the course of three rounds of biopanning.

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E. coli eCPX peptide

display

4 Incubation with bulk

metal target

Repeat screening with more

stringent wash conditions lo

increase affinity selection

Propagate bound cells

z

Screen for cell binders

to target, wash away

unbound cells

1

Isolate cells and sequence DNA to

Identilty peptides

ri

w

le.

le

to

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%.pn+ k.un/ I Iì.oM: k.unl RruM

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percentage of cells binding PA were observed through 3 rounds of biopanning (Fig. 2B). Such enrichment in target 

binders is typical of a successful biopanning technique. After the completion of biopanning, the final cell population is

cultured on plates in order to produce individual colonies. The peptide display library is genetically encoded in the cells 

and the identity of the enriched affinity peptides is easily determined using standard DNA sequencing techniques.

2.2 Nontraditional biopanning

The use of a protein target to develop affinity peptides from bacterial libraries may yield peptides useful in a variety of 

applications, as noted in the previous section. However, there exists application for peptide development using a bulk, 

macroscale target. The incorporation of a bulk target material into the biopanning technique requires critical 

modifications to the process and is therefore referred to as nontraditional biopanning. Most often the bulk target utilized 

in nontraditional biopanning is a metal and the developed affinity peptides have far reaching applications in various 

fields of study. Interest in these types of peptides has persisted for some time and phage display has been used 

extensively to this end. Peptide SRE’s have been developed with affinity to metals [34-39], oxides [40, 41], alloys [42], 

metal salts [43], and semiconductors [44-46] for use in the mineralization [47-49] and functionalization [45, 50] of 

surfaces. Some types of E. coli display systems have been used to develop metal compound binders. E. coli cell surface 

display has been utilized in conjunction a bulk metal target. Brown [13] developed an E. coli concatamer library 

displayed on the external domain of a lambda phage receptor and sorted for iron oxide binding.. The previously noted 

FimH library[14] was screened for zinc oxide binding and yielded several specific binding motifs. The E. coli flagellabased display system (FliTrx), developed by Lu et al. [7] has been used to sort for gold [15], silver [16], copper and zinc 

oxide [17] binders. 

Nontraditional biopanning with a bulk metal target precludes the use of automated, high throughput technologies like 

FACS due to the large size of the target (typically on the order of cm). Therefore, alternative methods must be developed 

for the selection and screening of affinity binders. A general schematic for nontraditional biopanning is shown in Fig.

3A. In traditional biopanning, the bacterial display library is cleaned and concentrated through centrifugation and 

resuspension in a buffer before the protein target is added. Steps may also be taken at this time to block nonspecific 

interactions by the addition of bovine serum albumin (BSA). In nontraditional biopanning, the bulk target is simply 

added to the library in the growth medium. Issues with nonspecific binding are address in the washing steps and no 

Figure 3. (A) Schematic diagram showing the steps involved in biopanning to bulk materials using a 

display library (B) Shows a typical enrichment in the % of a library population that binds over the course 

of four rounds of biopanning.

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blocking measures are taken during the incubation step. Also, incubation should be short (30 min or less) to promote 

specific and high affinity binding. Longer incubations may result in nonspecific attachment, such as early biofilm 

formation. Because target bound cells cannot be picked from the population, as is possible in traditional biopanning, the 

unbound cells must be efficiently removed and then the target bound cells isolated. With a bulk material, this is simply 

done by replenishing the media. Due to the importance of non-binder removal, the washing step is critical. A detergent, 

such as Tween20 or Triton X is often added to break non-specific attachments, and a vigorous agitation aids in the 

removal of loosely bound cells. By increasing the stringency of the wash conditions, the binder affinity can be enriched 

as the biopanning process progresses. As in traditional biopanning, the final steps consist of isolating individual colonies 

form the final biopanning round population and DNA sequencing analysis to identify the developed affinity peptides. 

Also similar to traditional biopanning, enrichment in cell binders to the target should be evident through the 

nontraditional biopanning process. We utilized the eCPX peptide display library to develop a nontraditional biopanning 

technique using a bulk aluminum alloy as a target material. Using a standard dilution plate count technique, we 

quantified the number of cells bound to the aluminum alloy surface after each round of biopanning. A clear trend in 

increasing cell attachment was observed, as shown in Fig. 3B. Although different from the traditional biopanning 

process utilizing nanoscale protein targets, successful affinity peptide development is possible using a nontraditional 

approach for bulk, macroscale targets, such as metal alloys.

3. ADVANCED BACTERIAL DISPLAY LIBRARIES

3.1 There is increasing interest in the development of affinity peptides f novel targets for a variety of applications 

in the research and development fields. The use of such targets will require biopanning approaches to be 

tailored to the specific target material and a cell surface display host capable of providing this level of 

flexibility will be necessary. E. coli is widely recognized for its relatively easy to manipulate genetic material 

that allows customized libraries to be generated and transformed into cells at very high efficiencies, both at a 

commercial scale and at the laboratory bench. Furthermore, the very rapid growth rate and simple culture 

requirement makes biodiscovery of novel peptides a relatively straightforward process that can be scaled-up 

for manufacturing. For these reasons, it is likely the use of E. coli as a cell surface display host and the new 

peptide libraries that utilize it will continue to expand, and it will play a role in the next generation of peptide 

reagent discovery. Survey of potential host E. coli strains

There are a number of commercially available E. coli host strains which have been used with phage display libraries, 

several of which can be purchased electrically competent, or ready to be transformed with a library. We obtained three of 

these electrically competent E. coli strains, ER2738, MC1061, and TG1 (Lucigen; Middlebrook, WI), and introduced the 

eCPX peptide display scaffold plasmid via electroporation according to manufacture instructions. After growing the 

transformed colonies on LB agar plates supplemented with 25μg/ mL chloramphenicol, the transformation efficiency for 

each strain was calculated using equation 1 and the results are displayed in Table 1.

(1)

All three strains transformed with relatively good efficiency, 107-108colony forming units (CFU)/ μg DNA, although the 

manufacture reported transformation efficiency of 4x109 CFU/μg with a pUC19 control plasmid was not obtained. The 

Strain pB33-eCPX Transformation 

Efficiency (CFU/μg)

ER2738 4 x 108

MC1061 3 x 107

TG1 2 x 108

Table 1. Table of transformation efficiencies from three host strains of E. coli.

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Positive Control. Strain

to2 lo' lo` 105 FITC-H Strain ER2 7 38

o-. U - 89%

2 nnnI I "nnI 1 ysio io io FITC-H

ó

U

Strain MC 1061

FITC-H

Strain TG 1

slightly lower efficiency is likely due to the size difference in the eCPX plasmid and the control pUC19 plasmid. 

Furthermore, the efficiency could likely be increased by optimizing the electroporation conditions specifically for the 

eCPX plasmid. The ability to effectively transform the host cell is critical for library construction because the ultimate 

library diversity can be negatively impacted by a poor transfer of the DNA library into the host cells, even if the DNA 

library is highly diverse. 

3.2 Evaluation of eCPX display ability

Each of the three E. coli strains described in Section 4.1 has key genetic features that make it amenable to the surface 

display of engineered proteins. In order for a strain to be used as a host for an advanced eCPX peptide display library, it 

must have both high transformation efficiency and be capable of good eCPX display scaffold expression on its cell 

surface. In order to evaluate the expression and display level of eCPX in each strain, flow cytometry with 25μL 250 nM 

YPet-Mona was used as previously described[26]. The results showed that MC1061 had the highest eCPX surface 

display of all three strains and was almost the same as the positive control (Fig. 4). ER2738 also had good eCPX surface 

display, however TG1 demonstrated very poor display. Based on these findings, E. coli strain MC1061 was determined 

to be the best host for the advanced eCPX library.

3.3 Real time cell reporting in advanced peptide libraries

As the use of biopanning continues with new, bulk material targets it becomes increasingly important to visualize and 

measure cell binding in real time and to facilitate high throughput screening capabilities currently found with protein 

targets. Whereas the protein target is typically tagged in traditional biopanning regimes, it is not feasible to label a bulk 

target. However, it possible to label the cells themselves such that binding to the target material can be monitored in real 

time. Studies are currently underway to genetically engineer the eCPX peptide display system to produce a stable 

fluorescent reporter within and allow cells to be visualized by using current fluorescence spectrophotometry

technologies. The commercially engineered red fluorescent protein, dsRed, is used in the current advanced library 

development, however another fluorescent protein can also be employed. The dsRed protein is very stable and is widely 

used in a variety of reporting-type applications and assays. The dsRed gene is inserted into the eCPX plasmid upstream 

Figure 4. Evaluation of eCPX bacterial library expression monitored via the YPET-Mona expression tag 

and fluorescence activated cell sorting (FACS) analysis. 

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B

of the eCPX-encoding gene, but under the same arabinose inducible promoter (Fig. 5A). The plasmid is then 

transformed into E. coli cells and co-expressed with the eCPX display scaffold. Cells which produce the dsRed protein 

are visible in the red region using confocal microscopy (Fig. 5B) and general UV light (Fig. 5C). Due to the high 

stability of dsRed, cells will remain fluorescent day after the protein is first produced and the fluorescence is not easily 

quenched. We believe that this is an ideal system for use in an advanced display library. Work is currently underway to 

produce a 15mer peptide library contain this dsRed reporter construct and examine its utility with nontraditional targets. 

This conceptualized real time cell reporter library would be incubated with a target of interest, like a bulk metal, 

unbound cells removed in a subsequent washing step, and the remaining bound cells visualized and/or their fluorescence 

intensity measured. The bound cells would then be propagated and additional biopanning rounds performed as described 

in section 3.2. The enrichment of target binding could be monitored in real time during the biopanning process and 

would be indicated by increasing fluorescence intensity. In addition to monitoring enrichment, the efficiency of the 

washing step could be monitored; a small change in fluorescence after washing would indicate an ineffective wash and 

an excessively large change may indicated an overly aggressive wash. This can be useful when developing a 

nontraditional biopanning approach for a new target. Additionally, both epifluorescence and confocal microscopy 

(depending on the target material) can be used to directly visualize bound cells on the material. Unlike electron 

microscopy techniques, the cells remain viable for downstream steps using these techniques.

4. CONCLUSION 

To conclude, we have previously shown the power of peptide discovery using bacterial display technology including 

rapid discovery with a low cost microfluidic sorting system. This work highlights our progress towards extending 

biopanning discovery of bacterial display libraries to peptide discovery for a wider range of material systems including 

bulk objects. Towards this end, advanced library development and clone analysis including integrated reporting will 

allow for simplified yet versatile discovery. The biopanning discovery for bulk materials using bacterial display offers a 

number of advantages with the most attractive being the rapid, simple discovery methodology empowered by the E. coli

host. Also, the ability to directly propagate the portion of the library that is bound to the sample without any 

complicated desorption steps which are likely to lead to loss of the best binding population is a desirable feature.

5. ACKNOWLEDGMENTS

The authors acknowledge the support of the US Army Research Laboratory, Sensors and Electron Devices Directorate 

(ARL-SEDD) and the ARL-SEDD SEED program for funding. Research is supported in part by Dr. Adams’ 

Figure 5. A) ds Red fluorescent protein gene insertion into the eCPX plasmid (B) epi-fluroescence microscopy 

image of integrated dsRed reporting with the e-CPX bacterial display library, and (C) UV illumination of two 

tubes of library stock, having no dsRed on the left, and integrated dsRed on the fluorescent image on the right 

acquired using a standard gel documentation system.

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appointment to the US Army Research Laboratory Postdoctoral Fellowship Program administered by the Oak Ridge 

Associated Universities through a contract with the US Army Research Laboratory. The authors would also like to 

acknowledge the talents of Mr. Eric Proctor and Mr. William Parks for the creation of the conceptual rendition of the 

unconstrained peptide bacterial display system. 

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