Evolutionary identification of affinity peptides for the detection of sepsis biomarker procalcitonin

Abstract

We demonstrate for the first time the use of phage display for identification and selection of novel peptides that are capable of binding to procalcitonin. The best peptide specific for procalcitonin has an amino acid sequence of ‘MSCAGHMCTRFV’ and the binding affinity was observed with a nanomolar binding.

---

Sepsis is a serious whole-body inflammation response which is caused by bacterial, viral or fungal infection and it thus remains the primary cause of death in spite of medical advances including the generation of new antibiotics or other chemical therapies.^1–4 Therefore, early detection and accurate diagnosis are important to achieve improved outcomes as well as the reduction of deaths. The use of microbial cultures has been regarded as gold standard method for prediction and detection of sepsis.^1,5 However, one of the major limitations in this approach is poor sensitive and long testing time during whole process.⁶ In ongoing efforts to address these bottlenecks, alternative diagnostic methods including PCR,¹ electrochemical immunoassay,⁶ and SPR⁵ have been demonstrated. Over several decades, some clinical biomarkers are commonly used for the early detection of sepsis including C-reactive protein (CRP), procalcitonin (ProCT), various cytokines and cell surface markers.^7,8 Among them, ProCT is ubiquitously and uniformly expressed in multiple tissues and the levels of ProCT are rapidly increased in serum within 12–48 h after bacterial invasion.^9,10 In addition, serum ProCT is more sensitive and specific as compared with other serum markers, for example, lactate, CRP and IL-6. Therefore, an elevated and circulating level of ProCTs have been widely investigated as a promising biomarker in the early diagnosis of sepsis.^3,11–13

Evolutionary phage display is a powerful method that allows for the identification of novel peptide motifs specific for targets of interest. Compared to antibody-based immunoassay, linear and short peptides have attracted to be effective recognition element for developing biosensing platforms.^14–16 One of the most exciting benefits of the peptides is small in size and cost-effective for the creation of new biosensors.¹⁴ More importantly, small peptides are less expensive production and lower immunogenicity.

In this study, we utilized phage display technique to screen novel peptides that are capable of binding to sepsis biomarker ProCT. After several rounds of biopanning, high affinity binding peptides were identified and characterized by ELISA assay. To our knowledge, this is the first example of identification and characterization of newly identified peptide binders for sepsis biomarker ProCT. In brief, the biotinylated recombinant human ProCTs were immobilized onto the streptavidin-coated microwell plate and washed with TBST after blocking. The Ph.D.-12 phage-displayed random peptide library (1.5 × 10¹³ pfu) was added into pre-coated well and the bound phages were eluted. After 5 rounds of biopanning, the peptides specific for ProCT were identified. The percent yields after 5 rounds biopanning were shown in Table S1 and S2.† As expected, the enrichment of the eluted phage clones was increased with the increase of biopanning. The samples of the eluted phages from each biopanning were used to analyze for DNA sequencing. While performing a biopanning process, we have chosen 3 interesting phage clones (3R#23 with amino acid sequence MSCAGHMCTRFV, 4R#22 with amino acid sequence QFDYMRPANDTH, 5R#24 with amino acid sequence AERVADHTVSVW) and measured the relative binding affinity for ProCT by ELISA. Fig. 1a demonstrated the binding affinity of the selected phages for ProCT proteins. Among the phage clones tested, 3R#23 showed the best peptides against their targets, compared to other clones. However, the binding affinities of 4R#22 and 5R#24 phage clones were quite low. Based on sequence analysis results, hydrophobic amino acids (Met, Cys, Ala, Phe and Val) were rich in 3R#23. This is noteworthy that the binding affinity of 3R#23 for BSA as control was little high, indicating that nonspecific binding or hydrophobic interactions, as shown in Fig. 1a. Especially, the appearance of hydrophobic (Met) and aromatic (Phe) may be involved in π–π and/or cation–π interactions and thus it may participate on the binding of ProCT.¹⁷ In addition, 2 cysteine residues at positions 3 and 8 were also found. Since 3R#23 contains even number of Cys, it may mediate on the formation of helical structure with S–S bond or multiple thioether bonds¹⁸ (Table S3†). Therefore, we have chosen 3R#23 phage clones as promising candidates for further experiments.

Fig. 1 ELISA assays. Selection of phage-displayed peptides specific for ProCT (a), the effects of phage concentration (b) and ProCT concentration (c) on binding affinity.

To see the effects of phage particle concentration on binding affinity, 3R#23 clones with different concentration ranging from 10⁵ to 10¹¹ pfu were incubated at 1.16 μM of ProCT protein and measured ELISA signal. Fig. 1b showed the relative binding property of 3R#23 clones at different phage particle concentrations. In the presence of 10¹¹ pfu mL⁻¹ of 3R#23 clones, the highest binding affinity was found. However, the affinity was sharply decreased below in that concentration, resulting in low binding to target proteins. This is possible due to the loss of avidity effects. As a consequence, we found that 3R#23 phage-displayed peptides specifically bind to ProCT. To further study the effects of ProCT concentration on binding affinity, approximately 10¹¹ pfu mL⁻¹ of 3R#23 phage clones were added into pre-immobilized well with ProCT protein in a variety of concentration from 0.06 to 1.16 μM and measured by ELISA (Fig. 1c). In fact, the binding affinity of 3R#23 clones was increased in a concentration-dependent manner. Interestingly, 3R#23 clones showed still strong binding affinity for ProCT protein as low as 0.06 μM. As expected, wild type M13 phages as control did not bind to ProCT (data not shown).

Fig. 2a showed the apparent binding constants (K_d,app) of the best performed peptides by in-direct ELISA assay. The K_d,app of the peptides was found to be 1.9 ± 0.001 nM affinity for ProCT. To further evaluate the effects of serum on binding affinity, 3R#23 clones in the presence of serum ranging from 0.1% to 1% were incubated at 1.16 μM of ProCT protein and measured ELISA signal. Fig. 2b showed the relative binding affinity of 3R#23 clones with or without serum. More interestingly, the peptides have still binding affinity to ProCT when spiked 0.1% of serum. However, the binding affinity was slightly decreased in the presence of 1% of serum.

Fig. 2 Apparent binding constants (K_d,app) (a) and the effects of serum on binding affinity (b).

Conclusions

In conclusion, novel peptide specific for sepsis biomarker ProCT was identified via phage display technique and characterized. The peptide specific for ProCT had a sequence of MSCAGHMCTRFV. ELISA assays were used to evaluate the binding interactions and the K_d,app value of the best performance peptide was found to be nanomolar affinity. In a serum spiking condition, the peptide was still binding to ProCT, even though binding affinity was gradually decreased in the presence of 1% of serum. As a consequence, we concluded that the free peptides away from phage particles or peptide-displayed phages may be useful for the detection of sepsis biomarker ProCT in real samples such as plasma or serum from sepsis patients. Although the phages used in this study can be generated approximately 5 copies of the peptide per phage, true K_d value of the free peptides for ProCT may be different due to the loss of the functional affinity. However, chemically or genetically modified phage particles can be used for the development of biosensors with improved binding affinity. The affinity of synthetic peptides and chemically modified phage particles compared with antibody is under consideration by using ITC, SPR as well as EIS.

Acknowledgements

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2014R1A2A2A01005621).

Notes and references

1. M. Kemmler, U. Sauer, E. Schleicher, C. Preininger and A. Brandenburg, Sens. Actuators, B, 2014, 192, 205–215.
2. J. Cohen, C. Brun-Buisson, A. Torres and J. Jorgensen, Crit. Care Med., 2004, 32, S466–S494.
3. T. Rowland, H. Hilliard, G. Barlow and S. M. Gregory, in Advances in Clinical Chemistry, Elsevier, 2015, vol. 68, pp. 71–86.
4. D. Anand, S. Das, S. Bhargava, L. M. Srivastava, A. Garg, N. Tyagi, S. Taneja and S. Ray, J. Crit. Care, 2015, 30, 218.
5. G. Sener, E. Ozgur, A. Y. Rad, L. Uzun, R. Say and A. Denizli, Analyst, 2013, 138, 6422–6428.
6. H. Li, Y. Sun, J. Elseviers, S. Muyldermans, S. Liu and Y. Wan, Analyst, 2014, 139, 3718–3721.
7. P. M. Kramer, Ke. E. Kremmer and S. Schulte-Hostede, Analyst, 2011, 136, 692–695.
8. S. Kibe, K. Adams and G. Barlow, J. Antimicrob. Chemother., 2011, 66, ii33–ii40.
9. B. M. Tang, G. D. Eslick, J. C. Craig and A. S. McLean, Lancet Infect. Dis., 2007, 7, 210–217.
10. A. A. Dahaba, B. Hagara, A. Fall, P. H. Rehak, W. F. List and H. Metzler, Br. J. Anaesth., 2006, 97, 503–508.
11. A. Viallon, F. Zeni, C. Lambert, B. Pozzetto, B. Tardy, C. Venet and J.-C. Bertrand, Clin. Infect. Dis., 1999, 28, 1310–1316.
12. L. Simon, F. Gauvin, D. K. Amre, P. Saint-Louis and J. Lacroix, Clin. Infect. Dis., 2004, 39, 206–217.
13. Y. Nakamura, A. Murai, M. Mizunuma, D. Ohta, Y. Kawano, N. Matsumoto, T. Nishida and H. Ishikura, J. Infect. Chemother., 2015, 21, 257–263.
14. H. J. Hwang, M. Y. Ryu and J. P. Park, RSC Adv., 2015, 5, 55300–55302.
15. S. T. Jung, K. J. Jeong, B. L. Iverson and G. Georgiou, Biotechnol. Bioeng., 2007, 98, 39–47.
16. J. Wu, J. P. Park, K. Dooley, D. M. Cropek, A. C. West and S. Banta, PLoS One, 2011, 6, e24948.
17. T. Sawada, Y. Okeya, M. Hashizume and T. Serizawa, Chem. Commun., 2013, 49, 5088–5090.
18. K. N. Enyedi, A. Czajlik, K. Knapp, A. Lang, Z. Majer, E. Lajko, L. Kohidai, A. Perczel and G. Mezo, J. Med. Chem., 2015, 58, 1806–1817.

---

Footnote

† Electronic supplementary information (ESI) available: Biotin labelling, biopanning and ELISA assay for measuring binding affinity. See DOI: 10.1039/c5ra20260d

---