, Volume 26, Issue 1, pp 113–122 | Cite as

Expression and characterization of recombinant bovine lactoferrin in E. coli

  • Isui García-Montoya
  • Jose Salazar-Martínez
  • Sigifredo Arévalo-Gallegos
  • Sugey Sinagawa-García
  • Quintin Rascón-Cruz


Lactoferrin is a member of the transferrin family of iron-binding proteins with a number of properties, including antibacterial activity against a broad spectrum of Gram-negative and Gram-positive bacteria. bovine lactoferrin cDNA was isolated, cloned and expressed as a fusion protein. The amino acid sequence of the fusion was analyzed and compared with other species. Crystallographic data were used to compare structural differences between bovine and human lactoferrin in 3-D models. A thioredoxin fusion protein was expressed and shown to have a different molecular weight compared with native bLf. After purification using Ni-NTA, the yield of recombinant bovine lactoferrin was 15.3 mg/l with a purity of 90.3 %. Recombinant bLf and pepsin-digested rbLf peptides demonstrated antibacterial activity of 79.8 and 86.9 %, respectively. The successful expression of functional, active and intact rbLf allows us to study the biochemical interactions of antimicrobial proteins and peptides and will facilitate their study as immunomodulators.


Bovine lactoferrin Antibacterial activity Thrombin Fusion expression Affinity chromatography 



This study was supported by internal resources found in the Universidad Autonoma de Chihuahua and Proteo/MuuTechnologies de Mexico.


  1. Adlerova L, Bartoskova A, Faldyna M (2008) Lactoferrin: a review. Vet Med 53:457–468Google Scholar
  2. Anderson BF, Baker HM, Dodson EJ, Norris GE, Rumball SV, Waters JM, Baker EN (1987) Structure of human lactoferrin at 3.2-A resolution. Proc Natl Acad Sci USA 84:1769–1773PubMedCrossRefGoogle Scholar
  3. Arnold K, Kiefer F, Kopp J, Battey JND, Podvinec M, Westbrook JD, Berman HM, Bordoli L, Schwede T (2009) The protein model portal. J Struct Funct Genomics 10:1–8PubMedCrossRefGoogle Scholar
  4. Baker EN, Baker HM (2005) Molecular structure, binding and dynamics of lactoferrin. Cell Mol Life Sci 62:2531–2539PubMedCrossRefGoogle Scholar
  5. Baker EN, Baker HM (2009) A structural framework for understanding the multifunctional character of lactoferrin. Biochimie 91:3–10PubMedCrossRefGoogle Scholar
  6. Bellamy W, Takase M, Wakabayashi H, Kawase K, Tomita (1992) Anti-bacterial spectrum of lactoferrin B, a potent bactericidal peptide derived from the N- terminal region of bovine lactoferrin. J App Bateriol 73:472–479CrossRefGoogle Scholar
  7. Box GEP, Behnken DW (1960) Some new three level designs for the study of quantitative variables. Technometrics 2:455–475CrossRefGoogle Scholar
  8. Brock J (2002) The physiology of lactoferrin. Biochem Cell Biol 80:1–6PubMedCrossRefGoogle Scholar
  9. Carpenter MA, Broad TE (1993) The cDNA sequence of horse transferrin. Biochim Biophys Acta 1173:230–232PubMedCrossRefGoogle Scholar
  10. Chapple DS, Mason DJ, Joannou CL, Odell EW, Gant V, Evans RW (1998) Structure–function relationship of antibacterial synthetic peptides homologous to a helical surface reginon human lactoferrin against Escherichia coli serotype O111. Infection Immun 66:2434–2440Google Scholar
  11. Choi BK, Actor JK, Rios S, d’anjou M, Stadheim TA, Warburton S, Giaccone E, Cukan M, Li H, Kull A, Sharkey N, Gollnick P, Kocieba M, Artym J, Zimecki M, Kruzel ML, Wildt S (2008) Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response. Glycoconj J 25:581–593PubMedCrossRefGoogle Scholar
  12. Cura V, Gangloff M, Elier S, Moras M, Ruff M (2007) Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ER in complex with synthetic ligands. Acta Crystallogr F 64:54–57CrossRefGoogle Scholar
  13. Elbarbary HA, Abdou AM, Young-Park Y, Nakamura Y, Mohamed HA, Sato K (2010) Novel antibacterial lactoferrin peptides generated by rennet digestion and autofocusing technique. Int Dairy Sci 20:646–651CrossRefGoogle Scholar
  14. Feng XJ, Wang JH, Shan AS, Teng D, Yang YL, Yao Y, Yang GP, Shao YC, Liu S, Zhang F (2006) Fusion expression of bovine lactoferricin in Eschericha coli. Protein Express Purif 47:110–117CrossRefGoogle Scholar
  15. Flores-Villaseñor H, Canizalez-Román A, Reyes-Lopez M, Mazmi K, de la Garza M, Zazueta-Beltrán J, León-Sicairos N, Bolscher JGM (2010) Bactericidal effect of bovine lactoferrin, LFcin, LFampin and LFchimera on antibiotic-resistant Staphylococcus aureus and Escherichia coli. Biometals 23:569–578PubMedCrossRefGoogle Scholar
  16. García-Montoya IA, Siqueiros-Cendon T, Arévalo-Gallegos S, Rascón-Cruz Q (2012) Lactoferrin a multiple bioactive protein: an overview. BBA-Gen Subjects 1820:226–236CrossRefGoogle Scholar
  17. Gonzalez-Chavez SA, Arévalo-Gallegos S, Rascón-Cruz Q (2009) Lactoferrin: structure, function and applications. Int J Antimicrob Agents 33:301PubMedCrossRefGoogle Scholar
  18. Jameson GB, Anderson BF, Norris GE, Thomas DH, Baker EN (1998) Structure of human apolactoferrin at 2.0 Å resolution. Refinement and analysis of ligand-induced conformational change. Acta Crystallogr D54:1319–1335Google Scholar
  19. Kim HK, Chun DS, Kim JS, Yun CH, Lee JH, Hong SK, Kang DK (2006) Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli. Appl Microbiol Biotechnol 72:330–338PubMedCrossRefGoogle Scholar
  20. Lee JH, Kim JH, Hwang SW, Lee WJ, Yoon HK, Lee HS, Hong SS (2000) High-level expression of antimicrobial peptide mediated by a fusion partner reinforcing formation of inclusion bodies. Biochem Biophys Res Commun 277:575–580PubMedCrossRefGoogle Scholar
  21. Lönnerdal B (2010) Bioactive proteins in human milk: mechanisms of action. J Pediatrics 156:S26–S30CrossRefGoogle Scholar
  22. Masson PL, Heremans FJ, Dive CH (1966) An iron-binding protein common to many external secretions. Clin Chim Acta 14:735–739CrossRefGoogle Scholar
  23. Montgomery DC (1997) Design and analysis of experiments. Response surface methods and other approaches to process optimization. Wiley, New York, pp 372–422Google Scholar
  24. Moore SA, Anderson BF, Groom CR, Haridas M, Baker EN (1997) Three-dimensional structure of diferric bovine lactoferrin at 2.8 angstrom resolution. J Mol Biol 274:222–236PubMedCrossRefGoogle Scholar
  25. Orsi N (2004) The antimicrobial activity of lactoferrin: current status and perspectives. Biometals 17:189–196PubMedCrossRefGoogle Scholar
  26. Park JH, Park GT, Cho IH, Sim SM, Yang JM, Lee DY (2011) An antimicrobial protein, lactoferrin exists in the sweat: proteomic analysis of sweat. Exp Dermatol 20:369–371PubMedCrossRefGoogle Scholar
  27. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera-a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612PubMedCrossRefGoogle Scholar
  28. Stark M, Liu L, Deber (2002) Cationic hydrophobic peptides with antimicrobial activity. Antimicrob Agents Chemother 46:3585–3590PubMedCrossRefGoogle Scholar
  29. Tian ZG, Teng D, Yang YL, Luo J, Feng XJ, Fan Y, Zhang F, Wang JH (2007) Multimerization and fusion expression of bovine lactoferricin derivative LfcinB15-W4, 10 in Escherichia coli. Appl Mircobiol Biotechnol 75:117–124CrossRefGoogle Scholar
  30. Tomita M, Bellamy W, Takase M, Yamauchi H, Kawase K (1991) Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin. J Dairy Sci 74:4137–4142PubMedCrossRefGoogle Scholar
  31. Tomita M, Wakabayashi H, Yamauchi Teraguchi S, Hayasawa H (2002) Bovine lactoferrin and lactoferricin derived from milk: production and applications. Biochem Cell Biol 80:109–112PubMedCrossRefGoogle Scholar
  32. van der Kraan MI, Groenink J, Nazmi K, Veerman EC, Bolscher JG, Nieuw Amerongen AV (2004) Lactoferrin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin. Peptides 25:177–183PubMedCrossRefGoogle Scholar
  33. Vogel HJ, Schibli DJ, Jing W, Lohmeier-Vogel EM, Epand RF, Epand RM (2002) Towards a structure–function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides. Biochem Cell Biol 80:49–63PubMedCrossRefGoogle Scholar
  34. Wally J, Buchanan SK (2007) A structural comparison of human serum transferrin and human lactoferrin. Biometals 20:249–262PubMedCrossRefGoogle Scholar
  35. Wang J, Tian Z, Teng D, Yang Y, Hu J, Wang J (2001) Cloning, expression and characterization of kunming lactoferrin and its N-Lobe. Biometals 23:523–530CrossRefGoogle Scholar
  36. Wright O, Yoshimi T, Tunnacliffe A (2012) Recombinant production of cathelicidin-derived antimicrobial peptides in Escherichia coli using an inducible autocleaving enzyme tag. New Biotechnol 29:352–358CrossRefGoogle Scholar
  37. Zarember KA, Sugui JA, Chang YC, Kwon-Chung KJ, Gallin JI (2007) Human polymorphonuclear leukocytes inhibits Aspergillus fumigatus conidial growth by lactoferrin mediated iron depletion. J Immunol 178:6367–6373PubMedGoogle Scholar
  38. Zhang H, Yoshida S, Aizawa T, Murakami R, Suzuki M, Koganezawa N, Matsuura A, Miyazawa M, Kawano K, Nitta K, Kato Y (2000) In vitro antimicrobial properties of recombinant ASABF, an antimicrobial peptide isolated from the nematode Ascaris suum. Antimicrob Agents Chemother 44:2701–2705PubMedCrossRefGoogle Scholar
  39. Zorko M, Jerala R (2010) Production of recombinant antimicrobial peptides in bacteria. Methods Mol Biol 618:61–76PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Isui García-Montoya
    • 1
  • Jose Salazar-Martínez
    • 2
  • Sigifredo Arévalo-Gallegos
    • 1
  • Sugey Sinagawa-García
    • 3
  • Quintin Rascón-Cruz
    • 1
  1. 1.Laboratorio de Biotecnología, Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Circuito 1Nuevo Campus UniversitarioChihuahuaMexico
  2. 2.Proteo/Muu-Technologies de MexicoGomez PalacioMexico
  3. 3.Facultad de Agronomía, Campus de Ciencias Agropecuarias, Universidad Autónoma de Nuevo LeónMonterreyMexico

Personalised recommendations