Expression and characterization of recombinant bovine lactoferrin in E. coli
- 877 Downloads
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.
KeywordsBovine 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.
- Adlerova L, Bartoskova A, Faldyna M (2008) Lactoferrin: a review. Vet Med 53:457–468Google Scholar
- 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
- 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
- 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
- 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
- Montgomery DC (1997) Design and analysis of experiments. Response surface methods and other approaches to process optimization. Wiley, New York, pp 372–422Google Scholar
- 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