Investigation of periplasmic signal peptides for the expression of Thanatin peptide in Escherichia coli

Elnaz Karbaschian ¹; Ali Javadmanesh ¹*

1, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

 

Manuscript link
http://dx.doi.org/10.30493/DLS.2021.475157

Abstract

The expression of periplasmic antimicrobial peptides is a critical aspect in cloning systems and protein expression. In the present study, bioinformatics methods were utilized to predict the optimal signal peptides for the expression of thanatin in Escherichia coli host. A total of 60 confirmed species-specific signal peptide sequences were obtained from signal peptide database. Since the aim of this study was to identify optimal signal peptides for periplasmic expression, the environment in which the protein operates, cleavage site, solubility, secretion probability, and the physical and chemical properties of the signal peptides were analyzed using SignalP6, SignalP4.1, Protein-Sol, and ProtParam. In the initial stage, 22 out of the 60 signal peptides passed through the SignalP6 and SignalP4.1 criteria. Solubility and secretion analysis of the signal peptides indicated that 22 signal peptides were suitable. Finally, the examination of physicochemical properties revealed that 21 signal peptides were the most suitable for the expression of thanatin. These final signal peptides were ranked by their D-Score. Among the top candidate signal peptides, E2BB, FMF4, PCOE, HSTI, FAEF were confirmed by other research.

Keywords: Signal peptides, Thanatin, Escherichia coli

References

1. Mancuso G, Midiri A, Gerace E, Biondo C. Bacterial antibiotic resistance: the most critical pathogens. Pathogens. 2021;10(10):1310. DOI
2. Rashidian Z, Roshanak S, Sekhavati MH, Javadmanesh A. Synergistic effects of nisin and CLF36 antimicrobial peptides in vitro. Vet. Res. Biol. Prod. 2023;36(3):44-50. DOI
3. Pokharel S, Shrestha P, Adhikari B. Antimicrobial use in food animals and human health: time to implement ‘One Health’ approach. Antimicrob. Resist. Infect. Control. 2020;9:1-5. DOI
4. Bobate S, Mahalle S, Dafale NA, Bajaj A. Emergence of environmental antibiotic resistance: Mechanism, monitoring and management. Environ. Adv. 2023;11:100409. DOI
5. Murray CJ, Ikuta KS, Sharara F, Swetschinski L, Aguilar GR, Gray A, Han C, Bisignano C, Rao P, Wool E, Johnson SC. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022;399(10325):629-55. DOI
6. Zhang QY, Yan ZB, Meng YM, Hong XY, Shao G, Ma JJ, Cheng XR, Liu J, Kang J, Fu CY. Antimicrobial peptides: mechanism of action, activity and clinical potential. Military Med. Res. 2021;8:1-25. DOI
7. Wang L, Wang N, Zhang W, Cheng X, Yan Z, Shao G, Wang X, Wang R, Fu C. Therapeutic peptides: current applications and future directions. Signal Transduct. Target Ther. 2022;7(1):48. DOI
8. Clark S, Jowitt TA, Harris LK, Knight CG, Dobson CB. The lexicon of antimicrobial peptides: a complete set of arginine and tryptophan sequences. Commun. Biol. 2021;4(1):605. DOI
9. Mousavi P, Morowvat MH, Mostafavi-Pour Z, Aram F, Malekzadeh K, Nezafat N, Ghasemi Y. Experimental analysis of E2BB (LTIIb) signal peptide in secretory production of reteplase in Escherichia coli. Int. J. Pept. Res. Ther. 2021;27:209-18. DOI
10. Vahedi F, Nassiri M, Ghovvati S, Javadmanesh A. Evaluation of different signal peptides using bioinformatics tools to express recombinant erythropoietin in mammalian cells. Int. J. Pept. Res. Ther. 2019;25:989-95. DOI
11. Khatami SH, Taheri-Anganeh M, Arianfar F, Savardashtaki A, Sarkari B, Ghasemi Y, Mostafavi-Pour Z. Analyzing signal peptides for secretory production of recombinant diagnostic antigen B8/1 from Echinococcus granulosus: an in silico approach. Mol. Biol. Res. Commun. 2020;9(1):1. DOI
12. Schlegel S, Rujas E, Ytterberg AJ, Zubarev RA, Luirink J, De Gier JW. Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels. Microb. Cell Fact. 2013;12:1-12. DOI
13. Forouharmehr A, Nassiri M, Ghovvati S, Javadmanesh A. Evaluation of different signal peptides for secretory production of recombinant bovine pancreatic ribonuclease A in gram-negative bacterial system: an in silico study. Curr. Proteomics. 2018;15(1):24-33. DOI
14. Kumari S, Chaurasia AK. In silico analysis and experimental validation of lipoprotein and novel Tat signal peptides processing in Anabaena sp. PCC7120. J. Microbiol. 2015;53:837-46. DOI
15. Javadmanesh A, Mohammadi E, Mousavi Z, Azghandi M, Tanhaiean A. Antibacterial effects assessment on some livestock pathogens, thermal stability and proposing a probable reason for different levels of activity of thanatin. Sci. Rep. 2021;11(1):10890. DOI
16. Hebditch M, Carballo-Amador MA, Charonis S, Curtis R, Warwicker J. Protein–Sol: a web tool for predicting protein solubility from sequence. Bioinformatics. 2017;33(19):3098-100. DOI
17. Ali F, Yao Z, Li W, Sun L, Lin W, Lin X. In-silico prediction and modeling of the quorum sensing LuxS protein and inhibition of AI-2 biosynthesis in Aeromonas hydrophila. Molecules. 2018;23(10):2627. DOI
18. Roshanak S, Yarabbi H, Shahidi F, Tabatabaei Yazdi F, Movaffagh J, Javadmanesh A. Effects of adding poly-histidine tag on stability, antimicrobial activity and safety of recombinant buforin I expressed in periplasmic space of Escherichia coli. Sci. Rep. 2023;13(1):5508. DOI
19. Teufel F, Almagro Armenteros JJ, Johansen AR, Gíslason MH, Pihl SI, Tsirigos KD, Winther O, Brunak S, von Heijne G, Nielsen H. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat. Biotechnol. 2022;40(7):1023-5. DOI
20. Choi JH, Lee S. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl. Microbiol. Biotechnol. 2004;64:625-35. DOI
21. Gasteiger E. Protein identification and analysis tools on the ExPASy server. In: The Proteomics Protocols Handbook. Humana Press. 2005. DOI
22. Byun H, Park J, Kim SC, Ahn JH. A lower isoelectric point increases signal sequence–mediated secretion of recombinant proteins through a bacterial ABC transporter. J. Biol. Chem. 2017;292(48):19782-91. DOI
23. 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;21:261-8. DOI
24. Auclair SM, Bhanu MK, Kendall DA. Signal peptidase I: Cleaving the way to mature proteins. Protein Sci. 2012;21(1):13–25. DOI

Cite this article:

Karbaschian E., Javadmanesh A.. Investigation of periplasmic signal peptides for the expression of Thanatin peptide in Escherichia coli. DYSONA–Life Science. 2025;6(1):7-16.