Analyzing Signal Peptides for Secretory Production of Recombinant Diagnostic Antigen B8/1 from Echinococcus granulosus: An In silico Approach

Document Type : Original article


1 Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

2 Cellular and Molecular Research Center, Research Institute on Cellular and Molecular Medicine, Urmia University of Medical Sciences,Urmia, Iran

3 Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran

4 Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran

5 Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

6 Department of Parasitology and Mycology School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

7 Basic Sciences in Infectious Diseases Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

8 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

9 Maternal-Fetal Medicine Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran


Recombinant AgB8/1 as the most evaluated antigen for serological diagnosis of Cystic Echinococcosis (CE) can provide early and accurate diagnosis for proper management and treatment of the disease. Thus, the secretory production of this recombinant protein is the main goal and the application of signal peptides at the N terminus of the desired protein can help to achieve this goal. The present study applied few bioinformatics tools to evaluate several signal peptides to offer the best candidate for extracellular production of AgB8/1 of Echinococcus granulosus in Escherichia coli. The sequences related to signal peptides were obtained from “Signal Peptide Website” and were checked by “UniProt”. In addition, UniProt was employed to retrieve the sequence of AgB8/1. Then, the probable signal peptide sequences and their cleavage site locations were determined by SignalP 4.1 followed by evaluation of their physicochemical features, using ProtParam. The solubility of the target recombinant proteins was accessed by SOLpro. Finally, PRED-TAT and ProtCompB were implemented to predict protein secretion pathways and final destinations. Among the 39 candidate signal peptides, ENTC2_STAAU and ENTC1_STAAU are the best ones which are stable and soluble in connection with AgB8/1 and can secrete target protein through Sec pathway. The signal peptides recommended in this investigation are valuable for rational designing of secretory stable and soluble AgB8/1. Such information is useful for future experimental production of the mentioned antigen.


1. Siracusano A, Margutti P, Delunardo F, Profumo E, Riganò R, Buttari B, Teggi A, Ortona E. Molecular cross-talk in host-parasite relationships: the intriguing immunomodulatory role of Echinococcus antigen B in cystic echinococcosis. Int J Parasitol 2008;38:1371-1376.
2. Torgerson P. Economic effects of echinococcosis. Acta Trop 2003;85:113-118.
3.Vola A, Tamarozzi F, Noordin R, Yunus MH, Khanbabaie S, De Silvestri A, Brunetti E, Mariconti M. Preliminary assessment of the diagnostic performances of a new rapid diagnostic test for the serodiagnosis of human cystic echinococcosis. Diagn Microbiol Infect Dis 2018;92:31-33.
4. Sarkari B, SFEDAN AF, Moshfe A, Khabisi SA, Savardashtaki A, Hosseini F, Shahbazi A. Clinical and molecular evaluation of a case of giant primary splenic hydatid cyst: A case report. Iran J Parasitol 2016;11:585.
5. Mohammadzadeh T, Sako Y, Sadjjadi SM, Sarkari B, Ito A. Comparison of the usefulness of hydatid cyst fluid, native antigen B and recombinant antigen B8/1 for serological diagnosis of cystic echinococcosis. Trans R Soc Trop Med Hyg 2012;106:371-375.
6. Siracusano A, Delunardo F, Teggi A, Ortona E. Cystic echinococcosis: aspects of immune response, immunopathogenesis and immune evasion from the human host. Endocr Metab Immune Disord Drug Targets 2012;12:16-23.
7. Manzano-Román R, Sánchez-Ovejero C, Hernández-González A, Casulli A, Siles-Lucas M. Serological diagnosis and follow-up of human cystic echinococcosis: a new hope for the future? Biomed Res Int 2015;2015.
8. Sarkari B, Rezaei Z. Immunodiagnosis of human hydatid disease: where do we stand? World J Methodol 2015;5:185.
9. Rott MB, Fernández V, Farias S, Ceni J, Ferreira HB, Haag KL, Zaha A. Comparative analysis of two different subunits of antigen B from Echinococcus granulosus: gene sequences, expression in Escherichia coli and serological evaluation. Acta Trop 2000;75: 331-340.
10. Virginio VG, Hernandez A, Rott MB, Monteiro KM, Zandonai AF, Nieto A, Ferreira HB. A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease. Clin Exp Immunol 2003;132:309-315.
11. Taheri-Anganeh M, Khatami SH, Jamali Z, Movahedpour A, Ghasemi Y, Savardashtaki A, Mostafavi-Pour Z. LytU-SH3b fusion protein as a novel and efficient enzybiotic against methicillin-resistant Staphylococcus aureus. Mol Biol Res Commun 2019:151-158.
12. Siles-Lucas M, Casulli A, Conraths F, Müller N. Laboratory diagnosis of Echinococcus spp. in human patients and infected animals.  Adv parasitol 2017;96:159-257.
13. Taheri-Anganeh M, Khatami SH, Jamali Z, Savardashtaki A, Ghasemi Y, Mostafavipour Z. In silico analysis of suitable signal peptides for secretion of a recombinant alcohol dehydrogenase with a key role in atorvastatin enzymatic synthesis. Mol Biol Res Commun 2019;8:17-26.
14. Bell MR, Engleka MJ, Malik A, Strickler JE. To fuse or not to fuse: what is your purpose? Protein Sci 2013;22:1466-1477.
15. Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 2014;5:172.
16. Kaur J, Kumar A, Kaur J. Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements. Int J Biol Macromol 2018;106:803-822.
17. Asadi M, Gharibi S, Khatami SH, Shabaninejad Z, Kargar F, Yousefi F, Taheri-Anganeh M, Savardashtaki A. Analysis of suitable signal peptides for designing a secretory thermostable cyanide degrading nitrilase: An in silico approach. J Environ Treat Tech 2019;7:506-513.
18. Zhong C, Wei P, Zhang YP. Enhancing functional expression of codon‐optimized heterologous enzymes in Escherichia coli BL21 (DE3) by selective introduction of synonymous rare codons. Biotechnol Bioeng 2017;114:1054-1064.
19. Owji H, Nezafat N, Negahdaripour M, Hajiebrahimi A, Ghasemi Y. A comprehensive review of signal peptides: Structure, roles, and applications. Eur J Cell Biol 2018;97:422-441.
20. Mousavi P, Mostafavi-Pour Z, Morowvat MH, Nezafat N, Zamani M, Berenjian A, Ghasemi Y. In silico analysis of several signal peptides for the excretory production of reteplase in Escherichia coli. Curr Proteomics 2017;14:326-335.
21. Chen L. Bioinformatics analysis of protein secretion in plants. Plant Protein Secretion: Springer; 2017. p.33-43.
22. Freudl R. Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact 2018;17:52.
23. Brockmeier U, Caspers M, Freudl R, Jockwer A, Noll T, Eggert T. Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. J Mol Biol 2006;362:393-402.
24. Chang CC, Song J, Tey BT, Ramanan RN. Bioinformatics approaches for improved recombinant protein production in Escherichia coli: protein solubility prediction. Brief Bioinform 2014;15:953-962.
25. Nielsen H. Predicting secretory proteins with SignalP. Protein Function Prediction: Methods Mol Biol 2017;1611:59-73.
26. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Marc R, Wilkins MR, Appel RD, Bairoch A. Protein identification and analysis tools on the ExPASy server.  The proteomics protocols Handbook: Springer; 2005.p.571-607.
27. Magnan CN, Randall A, Baldi P. SOLpro: accurate sequence-based prediction of protein solubility. Bioinformatics 2009;25:2200-2207.
28. Bagos PG, Nikolaou EP, Liakopoulos TD, Tsirigos KD. Combined prediction of Tat and Sec signal peptides with hidden Markov models. Bioinformatics 2010;26:2811-2817.
29. Zeng R, Gao S, Xu L, Liu X, Dai F. Prediction of pathogenesis-related secreted proteins from Stemphylium lycopersici. BMC Microbiol 2018;18:191.
30. Savardashtaki A, Sarkari B, Arianfar F, Mostafavi-Pour Z. Immunodiagnostic value of Echinococcus granulosus recombinant B8/1 subunit of antigen B. Iran J Immunol 2017;14: 111-122.
31. Negahdaripour M, Nezafat N, Hajighahramani N, Soheil Rahmatabadi S, Hossein Morowvat M, Ghasemi Y. In silico study of different signal peptides for secretory production of interleukin-11 in Escherichia coli. Curr Proteomics 2017;14:112-121.
32. Jia B, Jeon CO. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open Biol 2016;6:160196.
33. Zamani M, Nezafat N, Negahdaripour M, Dabbagh F, Ghasemi Y. In silico evaluation of different signal peptides for the secretory production of human growth hormone in E.coli. Int J Pept Res Ther 2015;21:261-268.
34. Reed B, Chen R. Biotechnological applications of bacterial protein secretion: from therapeutics to biofuel production. Res Microbiol 2013;164:675-682.
35. Rusch SL, Kendall DA. Interactions that drive Sec-dependent bacterial protein transport. Biochemistry 2007;46:9665-9673.
36. Green ER, Mecsas J. Bacterial secretion systems:an overview. Microbiol Spectr 2016;4.