Volume 9, Issue 3 (Vol.9 No.3 Oct 2020)                   rbmb.net 2020, 9(3): 348-356 | Back to browse issues page


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Rezaei S, Hadadian S, Khavari-Nejad R A, Norouzian D. Recombinant Tandem Repeated Expression of S3 and S∆3 Antimicrobial Peptides. rbmb.net. 2020; 9 (3) :348-356
URL: http://rbmb.net/article-1-557-en.html
Nano-Biotechnology Department, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran
Abstract:   (403 Views)
Background: Antimicrobial peptides (AMPs) are promising candidates for new generations of antibiotics to overcome the threats of multidrug-resistant infections as well as other industrial applications. Recombinant expression of small peptides is challenging due to low expression rates and high sensitivity to proteases. However, recombinant multimeric or fusion expression of AMPs facilitates cost-effective large-scale production of AMPs. In This project, S3 and S∆3 AMPs were expressed as fusion partners. S3 peptide is a 34 amino acid linear antimicrobial peptide derived from lipopolysaccharide (LPS) binding site of factor C of horseshoe crab hemolymph and S∆3 is a modified variant of S3 possessing more positive charges.

Methods: Two copy tandem repeat of the fusion protein (named as S∆3S3-2mer-GS using glycine- serine linker was expressed in E. coli. BL21 (DE3). After cell disruption and solubilization of inclusion bodies, the protein was purified by Ni -NTA affinity chromatography. Antimicrobial activity and cytotoxic properties of purified S∆3S3-2mer-GS were compared with a previously produced tetramer of S3 with the same glycine- serine linker (S3-4mer-GS) and each of monomeric blocks of S3 and S∆3.

Results: S∆3S3-2mer-GS was successfully expressed with an expression rate of 26%. The geometric average of minimum inhibitory concentration (MIC GM) of S∆3S3-2mer-GS was 28%, 34%, and 57% lower than S∆3, S3-4mer-GS, and S3, respectively. S∆3S3-2mer-GS had no toxic effect on eukaryotes human embryonic kidney cells at its MIC concentration.

Conclusions: tandem repeated fusion expression strategy could be employed as an effective technique for recombinant production of AMPs.
Full-Text [PDF 345 kb]   (87 Downloads)    
Type of Article: Original Article | Subject: Molecular Biology
Received: 2020/08/31 | Accepted: 2020/09/10 | Published: 2020/12/1

References
1. Pavithrra G, Rajasekaran R. Gramicidin Peptide to Combat Antibiotic Resistance: A Review. Int J Pept Res Ther. 2019:191-199. [DOI:10.1007/s10989-019-09828-0]
2. Nagarajan K, Marimuthu SK, Palanisamy S, Subbiah L. Peptide Therapeutics Versus Superbugs: Highlight on Current Research and Advancements. International Journal of Peptide Research and Therapeutics. 2018;24(1):19-33. [DOI:10.1007/s10989-017-9650-0]
3. Hancock RE, Haney EF, Gill EE. The immunology of host defence peptides: beyond antimicrobial activity. Nature Reviews Immunology. 2016;16(5):321-334. [DOI:10.1038/nri.2016.29] [PMID]
4. Marr AK, Gooderham WJ, Hancock REW. Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Curr Opin Pharmacol. 2006;6(5):468-72. [DOI:10.1016/j.coph.2006.04.006] [PMID]
5. Cirac A, Torné M, Badosa E, Montesinos E, Salvador P, Feliu L, et al. Rational Design of Cyclic Antimicrobial Peptides Based on BPC194 and BPC198. Molecules. 2017;22(7):1054. [DOI:10.3390/molecules22071054] [PMID] [PMCID]
6. Lyu Y, Yang Y, Lyu X, Dong N, Shan A. Antimicrobial activity, improved cell selectivity and mode of action of short PMAP-36-derived peptides against bacteria and Candida. scientific reports. 2016;6:27258. [DOI:10.1038/srep27258] [PMID] [PMCID]
7. Yin LM, Edwards MA, Li J, Yip CM, Deber CM. Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions. J Biol Chem. 2012;287(10):7738-7745. [DOI:10.1074/jbc.M111.303602] [PMID] [PMCID]
8. Dathe M, Nikolenko H, Meyer J, Beyermann M, Bienert M. Optimization of the antimicrobial activity of magainin peptides by modification of charge. FEBS Lett. 2001;501(2-3):146-50. [DOI:10.1016/S0014-5793(01)02648-5]
9. Tan NS, Ng MLP, Yau YH, Chong PKW, Ho B, Ding JL. Definition of endotoxin binding sites in horseshoe crab factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides. The FASEB Journal. 2000;14(12):1801-13. [DOI:10.1096/fj.99-0866com] [PMID]
10. Mina Sepahi, Ahangari Cohan, Shahin Hadadian, Dariush Norouzian. Effect of glutamic acid elimination/substitution on the biological activities of S3 cationic amphiphilic peptides. Prep Biochem Biotechnol. 2020;50(7):664-672. [DOI:10.1080/10826068.2020.1725772] [PMID]
11. Schmidtchen A, Pasupuleti M, Malmsten M. Effect of hydrophobic modifications in antimicrobial peptides. Advances in Colloid and Interface Science. 2014;205:265-274. [DOI:10.1016/j.cis.2013.06.009] [PMID]
12. Malmsten M, Kasetty G, Pasupuleti M, Alenfall J, Schmidtchen A. Highly selective end-tagged antimicrobial peptides derived from PRELP. PLoS One. 2011;6(1):e16400. [DOI:10.1371/journal.pone.0016400] [PMID] [PMCID]
13. Strömstedt AA, Pasupuleti M, Schmidtchen A, Malmsten M. Oligotryptophan-tagged antimicrobial peptides and the role of the cationic sequence. Biochim Biophys Acta. 2009;1788(9):1916-23. [DOI:10.1016/j.bbamem.2009.06.001] [PMID]
14. Schmidtchen A, Pasupuleti M, Mörgelin M, Davoudi M, Alenfall J, Chalupka A, et al. Boosting antimicrobial peptides by hydrophobic oligopeptide end tags. J Biol Chem. 2009;284(26):17584-94. [DOI:10.1074/jbc.M109.011650] [PMID] [PMCID]
15. Pasupuleti M, Schmidtchen A, Chalupka A, Ringstad L, Malmsten M. End-tagging of ultra-short antimicrobial peptides by W/F stretches to facilitate bacterial killing. PLoS One. 2009;4(4):e5285. [DOI:10.1371/journal.pone.0005285] [PMID] [PMCID]
16. Pasupuleti M, Chalupka A, Mörgelin M, Schmidtchen A, Malmsten M. Tryptophan end-tagging of antimicrobial peptides for increased potency against Pseudomonas aeruginosa. Biochim Biophys Acta. 2009;1790(8):800-8. [DOI:10.1016/j.bbagen.2009.03.029] [PMID]
17. Almaaytah A, Qaoud MT, Abualhaijaa A, Al-Balas Q, Alzoubi KH. Hybridization and antibiotic synergism as a tool for reducing the cytotoxicity of antimicrobial peptides. Infect Drug Resist. 2018;11:835-847. [DOI:10.2147/IDR.S166236] [PMID] [PMCID]
18. Ding JL, Zhu Y, Ho B. High-performance affinity capture-removal of bacterial pyrogen from solutions. Journal of Chromatography B: Biomedical Sciences and Applications. 2001;759(2):237-246. [DOI:10.1016/S0378-4347(01)00227-4]
19. Mina Sepahi SH, Reza Ahangari Cohan, Dariush Norouzian. Lipopolysaccharide removal affinity matrices based on novel cationic amphiphilic peptides. Preparative Biochemistry & Biotechnology. 2020. [DOI:10.1080/10826068.2020.1821216] [PMID]
20. Ding JL, Ho B, Tan NS. Recombinant proteins and peptides for endotoxin biosensors, endotoxin removal, and anti-microbial and anti- endotoxin therapeutics. US7297551 B2, United States Patent and Trademark Office, 2004.
21. Shtreimer Kandiyote N, Avisdris T, Arnusch CJ, Kasher R. Grafted Polymer Coatings Enhance Fouling Inhibition by an Antimicrobial Peptide on Reverse Osmosis Membranes. Langmuir. 2019;35(5):1935-43. [DOI:10.1021/acs.langmuir.8b03851] [PMID]
22. Li Y. Recombinant production of antimicrobial peptides in Escherichia coli: a review. Protein Expr Purif. 2011;80(2):260-7. [DOI:10.1016/j.pep.2011.08.001] [PMID]
23. Li Y. Carrier proteins for fusion expression of antimicrobial peptides in Escherichia coli. Biotechnol Appl Biochem. 2009;54(1):1-9. https://doi.org/10.1042/BA20090087 [DOI:10.1385/ABAB:125:1:001] [PMID] [PMCID]
24. Ding JL, Li P, Ho B. The Sushi peptides: structural characterization and mode of action against Gram-negative bacteria. Cell Mol Life Sci. 2008;65(7-8):1202-19. [DOI:10.1007/s00018-008-7456-0] [PMID]
25. Baghbeheshti S, Hadadian S, Eidi A, Pishkar L, Rahimi H. Effect of Flexible and Rigid Linkers on Biological Activity of Recombinant Tetramer Variants of S3 Antimicrobial Peptide. Int J Pept Res Ther. 2020. [DOI:10.1007/s10989-020-10095-7]
26. Yau YH, Ho B, Tan NS, Ng ML, Ding JL. High therapeutic index of factor C Sushi peptides: potent antimicrobials against Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2001;45(10):2820-5. [DOI:10.1128/AAC.45.10.2820-2825.2001] [PMID] [PMCID]
27. Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD, Bairoch A. Protein identification and analysis tools on the ExPASy server. The proteomics protocols handbook. 2005:571-607. [DOI:10.1385/1-59259-890-0:571]
28. He F. Laemmli-sds-page. Bio-protocol. 2011;1(11):1-4. [DOI:10.21769/BioProtoc.80]
29. Liu Z-Q, Mahmood T, Yang P-C. Western blot: technique, theory and trouble shooting. N Am J Med Sci. 2014;6(3):160. [DOI:10.4103/1947-2714.128482] [PMID] [PMCID]
30. ProtParam E. ExPASy-ProtParam tool. 2017.
31. Smith SM, Beattie AJ, Gillings MR, Holley MP, Stow AJ, Turnbull CL, et al. An enhanced miniaturized assay for antimicrobial prospecting. J Microbiol Methods. 2008;72(1):103-6. [DOI:10.1016/j.mimet.2007.10.003] [PMID]
32. Lee J, Kim J, Hwang S, Lee W, Yoon H, Lee H, et al. High-level expression of antimicrobial peptide mediated by a fusion partner reinforcing formation of inclusion bodies. Biochem Biophys Res Commun. 2000;277(3):575-80. https://doi.org/10.1006/bbrc.2000.3712 [DOI:10.1006/bbrc.1996.0445] [PMID]

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