Applied Microbiology and Biotechnology

, Volume 103, Issue 2, pp 659–671 | Cite as

Recent achievements and perspectives for large-scale recombinant production of antimicrobial peptides

  • David WibowoEmail author
  • Chun-Xia ZhaoEmail author


Antibiotic resistance poses a growing threat to global public health. It is urgent to develop new alternative antibiotics. Antimicrobial peptide (AMP) is a diverse class of natural-occurring molecules that constitute immune systems of living organisms. More than 2500 AMPs have been identified and isolated from natural sources. Compared to conventional antibiotics, AMPs exhibit antimicrobial activities against a broad spectrum of microorganisms including bacteria, fungi, and even viruses. More importantly, the unique antimicrobial mechanisms of AMPs make it difficult for microorganisms to develop resistance. Therefore, it is very promising to develop AMPs as high-value antimicrobial candidates. This mini review provides an update of recent progresses in recombinant production of AMPs after fusion of AMP with carrier proteins and their scale-up. Key factors including selection of expression host and fusion tags are firstly introduced, followed by subsequent discussions on purification of fusion proteins and recovery of antimicrobial peptides. The scale production of AMPs is also explored.


Antimicrobial peptides Fusion proteins Protein purification Recombinant production 


Compliance with ethical standards

Conflict of interests

The authors declare that they have no financial or commercial conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Abou Aleinein R, Hamoud R, Schafer H, Wink M (2013) Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities. Appl Microbiol Biotechnol 97(8):3535–3543. Google Scholar
  2. Arnau J, Lauritzen C, Petersen GE, Pedersen J (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48(1):1–13. Google Scholar
  3. Azevedo AM, Rosa PAJ, Ferreira IF, Aires-Barros MR (2009) Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing. Trends Biotechnol 27(4):240–247. Google Scholar
  4. Bahar A, Ren D (2013) Antimicrobial peptides. Pharmaceuticals 6(12):1543., 1575Google Scholar
  5. Bell MR, Engleka MJ, Malik A, Strickler JE (2013) To fuse or not to fuse: what is your purpose? Protein Sci 22(11):1466–1477. Google Scholar
  6. Bray BL (2003) Large-scale manufacture of peptide therapeutics by chemical synthesis. Nat Rev Drug Discov 2:587–593. Google Scholar
  7. Cao J, de la Fuente-Nunez C, Ou RW, Torres MDT, Pande SG, Sinskey AJ, Lu TK (2018) Yeast-based synthetic biology platform for antimicrobial peptide production. ACS Synth Biol 7(3):896–902. Google Scholar
  8. Casciaro B, Dutta D, Loffredo MR, Marcheggiani S, McDermott AM, Willcox MD, Mangoni ML (2018) Esculentin-1a derived peptides kill Pseudomonas aeruginosa biofilm on soft contact lenses and retain antibacterial activity upon immobilization to the lens surface. Biopolymers 110(5):e23074. Google Scholar
  9. Che YY, Lu YH, Zha XD, Huang HQ, Yang PL, Ma LJ, Xu XJ (2016) Higher efficiency soluble prokaryotic expression, purification, and structural analysis of antimicrobial peptide G13. Protein Expr Purif 119:45–50. Google Scholar
  10. Chen X, Zaro JL, Shen W-C (2013) Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 65(10):1357–1369. Google Scholar
  11. Chen X, Shi JW, Chen R, Wen YA, Shi Y, Zhu Z, Guo SW, Li L (2015) Molecular chaperones (TrxA, SUMO, Intein, and GST) mediating expression, purification, and antimicrobial activity assays of plectasin in Escherichia coli. Biotechnol Appl Biochem 62(5):606–614. Google Scholar
  12. Chen X, Li J, Sun H, Li S, Chen T, Liu G, Dyson P (2017) High-level heterologous production and functional secretion by recombinant Pichia pastoris of the shortest proline-rich antibacterial honeybee peptide apidaecin. Sci Rep 7(1):14543. Google Scholar
  13. Chopra L, Singh G, Choudhary V, Sahoo DK (2014) Sonorensin: an antimicrobial peptide, belonging to the heterocycloanthracin subfamily of bacteriocins, from a new marine isolate, Bacillus sonorensis MT93. Appl Environ Microbiol 80(10):2981–2990. Google Scholar
  14. Corsini L, Hothorn M, Scheffzek K, Sattler M, Stier G (2008) Thioredoxin as a fusion tag for carrier-driven crystallization. Protein Sci 17(12):2070–2079. Google Scholar
  15. Deng T, Ge H, He H, Liu Y, Zhai C, Feng L, Yi L (2017) The heterologous expression strategies of antimicrobial peptides in microbial systems. Protein Expr Purif 140:52–59. Google Scholar
  16. Dimitrijev Dwyer M, Brech M, Yu L, Middelberg APJ (2014) Intensified expression and purification of a recombinant biosurfactant protein. Chem Eng Sci 105:12–21. Google Scholar
  17. Dutta D, Kamphuis B, Ozcelik B, Thissen H, Pinarbasi R, Kumar N, Willcox MDP (2018) Development of silicone hydrogel antimicrobial contact lenses with Mel4 peptide coating. Optom Vis Sci 95(10):937–946. Google Scholar
  18. Eckert R (2011) Road to clinical efficacy: challenges and novel strategies for antimicrobial peptide development. Future Microbiol 6(6):635–651. Google Scholar
  19. Enfors SO, Jahic M, Rozkov A, Xu B, Hecker M, Jürgen B, Krüger E, Schweder T, Hamer G, O'Beirne D, Noisommit-Rizzi N, Reuss M, Boone L, Hewitt C, McFarlane C, Nienow A, Kovacs T, Trägårdh C, Fuchs L, Revstedt J, Friberg PC, Hjertager B, Blomsten G, Skogman H, Hjort S, Hoeks F, Lin HY, Neubauer P, van der Lans R, Luyben K, Vrabel P, Manelius Å (2001) Physiological responses to mixing in large scale bioreactors. J Biotechnol 85(2):175–185. Google Scholar
  20. Feng XJ, Xu WS, Qu P, Li XC, Xing LW, Liu D, Jiao J, Wang J, Li ZQ, Liu CL (2015) High-yield recombinant expression of the chicken antimicrobial peptide fowlicidin-2 in Escherichia coli. Biotechnol Prog 31(2):369–374. Google Scholar
  21. Ganz T (2003) The role of antimicrobial peptides in innate immunity. Integr Comp Biol 43(2):300–304. Google Scholar
  22. Gaspar D, Veiga AS, Castanho MARB (2013) From antimicrobial to anticancer peptides. A review. Front Microbiol 4(294).
  23. Gibbs GM, Davidson BE, Hillier AJ (2004) Novel expression system for large-scale production and purification of recombinant class IIa bacteriocins and its application to piscicolin 126. Appl Environ Microbiol 70(6):3292–3297. Google Scholar
  24. Harder J, Bartels J, Christophers E, Schröder JM (1997) A peptide antibiotic from human skin. Nature 387:861. Google Scholar
  25. Herbel V, Schafer H, Wink M (2015) Recombinant production of snakin-2 (an antimicrobial peptide from tomato) in E-coli and analysis of its bioactivity. Molecules 20(8):14889–14901. Google Scholar
  26. Hou HH, Yan WL, Du KX, Ye YJ, Cao QQ, Ren WH (2013) Construction and expression of an antimicrobial peptide scolopin 1 from the centipede venoms of Scolopendra subspinipes mutilans in Escherichia coli using SUMO fusion partner. Protein Expr Purif 92(2):230–234. Google Scholar
  27. Huang C-J, Lin H, Yang X (2012) Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. J Ind Microbiol Biotechnol 39(3):383–399. Google Scholar
  28. Hunt I (2005) From gene to protein: a review of new and enabling technologies for multi-parallel protein expression. Protein Expr Purif 40(1):1–22. Google Scholar
  29. Ji S, Li W, Baloch AR, Wang M, Li H, Cao B, Zhang H (2017) Efficient biosynthesis of a cecropin A-melittin mutant in Bacillus subtilis WB700. Sci Rep 7:40587. Google Scholar
  30. Klint JK, Senff S, Saez NJ, Seshadri R, Lau HY, Bende NS, Undheim EAB, Rash LD, Mobli M, King GF (2013) Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli. PLoS One 8(5):e63865. Google Scholar
  31. Latham PW (1999) Therapeutic peptides revisited. Nat Biotechnol 17:755–757. Google Scholar
  32. Lee SY (1996) High cell-density culture of Escherichia coli. Trends Biotechnol 14(3):98–105. Google Scholar
  33. Li Y (2010) Carrier proteins for fusion expression of antimicrobial peptides in Escherichia coli. Biotechnol Appl Biochem 54(1):1–9. Google Scholar
  34. Li Y (2011) Recombinant production of antimicrobial peptides in Escherichia coli: a review. Protein Expr Purif 80(2):260–267. Google Scholar
  35. Li YF (2013a) Production of human antimicrobial peptide LL-37 in Escherichia coli using a thioredoxin-SUMO dual fusion system. Protein Expr Purif 87(2):72–78. Google Scholar
  36. Li YF (2013b) Recombinant production of crab antimicrobial protein scygonadin expressed as thioredoxin and SUMO fusions in Escherichia coli. Appl Biochem Biotechnol 169(6):1847–1857. Google Scholar
  37. Li X, Leong SSJ (2011) A chromatography-focused bioprocess that eliminates soluble aggregation for bioactive production of a new antimicrobial peptide candidate. J Chromatogr A 1218(23):3654–3659. Google Scholar
  38. Li P, Li X, Saravanan R, Li CM, Leong SSJ (2012) Antimicrobial macromolecules: synthesis methods and future applications. RSC Adv 2(10):4031–4044. Google Scholar
  39. Li Y, Wang J, Yang J, Wan C, Wang X, Sun H (2014) Recombinant expression, purification and characterization of antimicrobial peptide ORBK in Escherichia coli. Protein Expr Purif 95:182–187. Google Scholar
  40. Lin CH, Pan YC, Liu FW, Chen CY (2017) Prokaryotic expression and action mechanism of antimicrobial LsGRP1C recombinant protein containing a fusion partner of small ubiquitin-like modifier. Appl Microbiol Biotechnol 101(22):8129–8138. Google Scholar
  41. Luan C, Zhang HW, Song DG, Xie YG, Feng J, Wang YZ (2014) Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology. Appl Microbiol Biotechnol 98(8):3651–3658. Google Scholar
  42. Mai S, Mauger MT, Niu L-N, Barnes JB, Kao S, Bergeron BE, Ling J-Q, Tay FR (2017) Potential applications of antimicrobial peptides and their mimics in combating caries and pulpal infections. Acta Biomater 49:16–35. Google Scholar
  43. Mangoni ML, McDermott AM, Zasloff M (2016) Antimicrobial peptides and wound healing: biological and therapeutic considerations. Exp Dermatol 25(3):167–173. Google Scholar
  44. Meng D-M, Dai H-X, Gao X-F, Zhao J-F, Guo Y-J, Ling X, Dong B, Zhang Z-Q, Fan Z-C (2016a) Expression, purification and initial characterization of a novel recombinant antimicrobial peptide Mytichitin-A in Pichia pastoris. Protein Expr Purif 127:35–43. Google Scholar
  45. Meng FQ, Zhao HZ, Zhang C, Lu FX, Bie XM, Lu ZX (2016b) Expression of a novel bacteriocin-the plantaricin Pln1-in Escherichia coli and its functional analysis. Protein Expr Purif 119:85–93. Google Scholar
  46. Meng D-M, Zhao J-F, Ling X, Dai H-X, Guo Y-J, Gao X-F, Dong B, Zhang Z-Q, Meng X, Fan Z-C (2017) Recombinant expression, purification and antimicrobial activity of a novel antimicrobial peptide PaDef in Pichia pastoris. Protein Expr Purif 130:90–99. Google Scholar
  47. Merrifield RB (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc 85(14):2149–2154. Google Scholar
  48. Middelberg APJ (2012) Releasing biopharmaceutical products from cells. In: Subramanian G (ed) Biopharmaceutical production technology, vol 1. Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimGoogle Scholar
  49. Mishra B, Reiling S, Zarena D, Wang G (2017) Host defense antimicrobial peptides as antibiotics: design and application strategies. Curr Opin Chem Biol 38:87–96. Google Scholar
  50. Müller H, Salzig D, Czermak P (2015) Considerations for the process development of insect-derived antimicrobial peptide production. Biotechnol Prog 31(1):1–11. Google Scholar
  51. Nguyen LT, Haney EF, Vogel HJ (2011) The expanding scope of antimicrobial peptide structures and their modes of action. Trends Biotechnol 29(9):464–472. Google Scholar
  52. Nordström R, Malmsten M (2017) Delivery systems for antimicrobial peptides. Adv Colloid Interf Sci 242:17–34. Google Scholar
  53. Onaizi SA, Leong SSJ (2011) Tethering antimicrobial peptides: current status and potential challenges. Biotechnol Adv 29(1):67–74. Google Scholar
  54. Pal G, Srivastava S (2015) Scaling up the production of recombinant antimicrobial plantaricin E from a heterologous host, Escherichia coli. Probiotics Antimicrob Proteins 7(3):216–221. Google Scholar
  55. Panteleev PV, Ovchinnikova TV (2017) Improved strategy for recombinant production and purification of antimicrobial peptide tachyplesin I and its analogs with high cell selectivity. Biotechnol Appl Biochem 64(1):35–42. Google Scholar
  56. Pina AS, Lowe CR, Roque ACA (2014) Challenges and opportunities in the purification of recombinant tagged proteins. Biotechnol Adv 32(2):366–381. Google Scholar
  57. Qu H, Chen B, Peng H, Wang K (2013) Molecular cloning, recombinant expression, and antimicrobial activity of EC-hepcidin3, a new four-cysteine hepcidin isoform from Epinephelus coioides. Biosci Biotechnol Biochem 77(1):103–110. Google Scholar
  58. Rezaei Javan R, van Tonder AJ, King JP, Harrold CL, Brueggemann AB (2018) Genome sequencing reveals a large and diverse repertoire of antimicrobial peptides. Front Microbiol 9(2012).
  59. Richard C, Drider D, Elmorjani K, Marion D, Prévost H (2004) Heterologous expression and purification of active divercin V41, a class IIa bacteriocin encoded by a synthetic gene in Escherichia coli. J Bacteriol 186(13):4276–4284. Google Scholar
  60. Schaller A, Connors NK, Dwyer MD, Oelmeier SA, Hubbuch J, Middelberg APJ (2015) Computational study of elements of stability of a four-helix bundle protein biosurfactant. J Comput Aided Mol Des 29(1):47–58. Google Scholar
  61. Schmidt FR (2005) Optimization and scale up of industrial fermentation processes. Appl Microbiol Biotechnol 68(4):425–435. Google Scholar
  62. Schreiber C, Müller H, Birrenbach O, Klein M, Heerd D, Weidner T, Salzig D, Czermak P (2017) A high-throughput expression screening platform to optimize the production of antimicrobial peptides. Microb Cell Factories 16(1):29. Google Scholar
  63. Song D, Chen Y, Li X, Zhu M, Gu Q (2014) Heterologous expression and purification of dermaseptin S4 fusion in Escherichia coli and recovery of biological activity. Prep Biochem Biotechnol 44(6):598–607. Google Scholar
  64. Sørensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115(2):113–128. Scholar
  65. Sousa DA, Mulder KCL, Nobre KS, Parachin NS, Franco OL (2016) Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP-intein tag. J Biotechnol 234:83–89. Google Scholar
  66. Sun Y, Li Q, Li Z, Zhang Y, Zhao J, Wang L (2012) Molecular cloning, expression, purification, and functional characterization of palustrin-2CE, an antimicrobial peptide of Rana chensinensis. Biosci Biotechnol Biochem 76(1):157–162. Google Scholar
  67. Sun B, Wibowo D, Middelberg APJ, Zhao C-X (2018a) Cost-effective downstream processing of recombinantly produced pexiganan peptide and its antimicrobial activity. AMB Express 8(1):6. Google Scholar
  68. Sun B, Wibowo D, Sainsbury F, Zhao C-X (2018b) Design and production of a novel antimicrobial fusion protein in Escherichia coli. Appl Microbiol Biotechnol 102(20):8763–8772. Google Scholar
  69. Tao Y, Zhao DM, Wen Y (2014) Expression, purification and antibacterial activity of the channel catfish hepcidin mature peptide. Protein Expr Purif 94:73–78. Google Scholar
  70. Tareq FS, Kim JH, Lee MA, Lee H-S, Lee J-S, Lee Y-J, Shin HJ (2013) Antimicrobial gageomacrolactins characterized from the fermentation of the marine-derived bacterium Bacillus subtilis under optimum growth conditions. J Agric Food Chem 61(14):3428–3434. Google Scholar
  71. Tay DKS, Rajagopalan G, Li X, Chen Y, Lua LHL, Leong SSJ (2010) A new bioproduction route for a novel antimicrobial peptide. Biotechnol Bioeng 108(3):572–581. Google Scholar
  72. Toennies G, Homiller RP (1942) The oxidation of amino acids by hydrogen peroxide in formic acid. J Am Chem Soc 64(12):3054–3056. Google Scholar
  73. Travkova OG, Moehwald H, Brezesinski G (2017) The interaction of antimicrobial peptides with membranes. Adv Colloid Interf Sci 247:521–532. Google Scholar
  74. Urry DW, Trapane TL, Prasad KU (1985) Phase-structure transitions of the elastin polypentapeptide–water system within the framework of composition–temperature studies. Biopolymers 24(12):2345–2356. Google Scholar
  75. Ventola CL (2015) The antibiotic resistance crisis: part 1: causes and threats. P & T 40(4):277–283Google Scholar
  76. Vu TTT, Jeong B, Yu J, Koo B-K, Jo S-H, Robinson RC, Choe H (2014) Soluble prokaryotic expression and purification of crotamine using an N-terminal maltose-binding protein tag. Toxicon 92:157–165. Google Scholar
  77. Wang XJ, Wang XM, Teng D, Zhang Y, Mao RY, Wang JH (2014) Recombinant production of the antimicrobial peptide NZ17074 in Pichia pastoris using SUMO3 as a fusion partner. Lett Appl Microbiol 59(1):71–78. Google Scholar
  78. Wang G, Li X, Wang Z (2016) APD3: the antimicrobial peptide database as a tool for research and education. Nucleic Acids Res 44(D1):D1087–D1093. Google Scholar
  79. Wei XB, Wu RJ, Zhang LL, Ahmad B, Si DY, Zhang RJ (2018) Expression, purification, and characterization of a novel hybrid peptide with potent antibacterial activity. Molecules 23(6).
  80. Weuster-Botz D (2000) Experimental design for fermentation media development: statistical design or global random search? J Biosci Bioeng 90(5):473–483. Google Scholar
  81. Wibowo D, Zhao C-X, Middelberg APJ (2015) Interfacial biomimetic synthesis of silica nanocapsules using a recombinant catalytic modular protein. Langmuir 31(6):1999–2007. Google Scholar
  82. Wibowo D, Yang G-Z, Middelberg APJ, Zhao C-X (2017) Non-chromatographic bioprocess engineering of a recombinant mineralizing protein for the synthesis of silica nanocapsules. Biotechnol Bioeng 114(2):335–343. Google Scholar
  83. Winkler DFH, Tian K (2015) Investigation of the automated solid-phase synthesis of a 38mer peptide with difficult sequence pattern under different synthesis strategies. Amino Acids 47(4):787–794. Google Scholar
  84. Wood DW, Wu W, Belfort G, Derbyshire V, Belfort M (1999) A genetic system yields self-cleaving inteins for bioseparations. Nat Biotechnol 17:889–892. Google Scholar
  85. Xie YG, Luan C, Zhang HW, Han FF, Feng J, Choi YJ, Groleau D, Wang YZ (2013) Effects of thioredoxin: SUMO and intein on soluble fusion expression of an antimicrobial peptide OG2 in Escherichia coli. Protein Pept Lett 20(1):54–60. Google Scholar
  86. Yadav DK, Yadav N, Yadav S, Haque S, Tuteja N (2016) An insight into fusion technology aiding efficient recombinant protein production for functional proteomics. Arch Biochem Biophys 612:57–77. Google Scholar
  87. Yee L, Blanch HW (1993) Defined media optimization for growth of recombinant Escherichia coli X90. 41(2):221–230.
  88. Yi TH, Sun SY, Huang YB, Chen YX (2015) Prokaryotic expression and mechanism of action of alpha-helical antimicrobial peptide A20L using fusion tags. BMC Biotechnol 15:69. Google Scholar
  89. Young CL, Britton ZT, Robinson AS (2012) Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J 7(5):620–634. Google Scholar
  90. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395. Google Scholar
  91. Zhang C, He XL, Gu YP, Zhou HY, Cao J, Gao Q (2014) Recombinant scorpine produced using SUMO fusion partner in Escherichia coli has the activities against clinically isolated bacteria and inhibits the Plasmodium falciparum parasitemia in vitro. PLoS One 9(7):e103456. Google Scholar
  92. Zhang LC, Li XD, Wei DD, Wang J, Shan AS, Li ZY (2015a) Expression of plectasin in Bacillus subtilis using SUMO technology by a maltose-inducible vector. J Ind Microbiol Biotechnol 42(10):1369–1376. Google Scholar
  93. Zhang Y, Teng D, Wang X, Mao R, Cao X, Hu X, Zong L, Wang J (2015b) In vitro and in vivo characterization of a new recombinant antimicrobial peptide, MP1102, against methicillin-resistant Staphylococcus aureus. Appl Microbiol Biotechnol 99(15):6255–6266. Google Scholar
  94. Zhao C-X, Dwyer MD, Yu AL, Wu Y, Fang S, Middelberg APJ (2015) A simple and low-cost platform technology for producing pexiganan antimicrobial peptide in E. coli. Biotechnol Bioeng 112(5):957–964. Google Scholar
  95. Zydney AL (2016) Continuous downstream processing for high value biological products: a review. Biotechnol Bioeng 113(3):465–475. Google Scholar

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© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Australian Institute for Bioengineering and NanotechnologyThe University of QueenslandSt LuciaAustralia
  2. 2.Griffith Institute for Drug DiscoveryGriffith UniversityNathanAustralia

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