Design of a recombinant bovine lactoferricin peptide and its expression in Pichia pastoris

  • LYU Zili ,
  • ZHANG Enpeng ,
  • GUO Aizhen ,
  • LUO Bin ,
  • LIANG Xin ,
  • SHAN Xudong ,
  • ZHUANG Jing ,
  • ZHANG Xia ,
  • WANG Liang
Expand
  • 1College of Medicine and Life Science, Chengdu University of TMC, Chengdu 610041, China
    2College of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013,China

Online published: 2019-09-03

Abstract

A LfcinB-derived peptide LfcinB-W4,10 was designed based on the structure-function relationship of antimicrobial peptides based on antimicrobial peptide database and bioinformatics analysis. In order to verify the rationality of the designed derivative peptide and the functional correctness of the expression product, the optimized gene sequence was digested with restriction endonucleases and ligated into the secretory expression vector pPIC9K with T4 DNA ligase. The recombinant expression vector pPIC9K-LFcinB-W4,10 was linearized by Sac I and transformed into Pichia pastoris GS115 by electroporation, and G418 was used to obtain high copy transformants. The results showed the target gene was stably integrated with the yeast genome. The expression of positive transformants were induced by 2.5% (v/v) methanol, and the expressed product was LfcinB-W4,10 with a relative molecular weight of 3.2 kDa detected by Tricine-SDS-PAGE. Moreover, the antibacterial activity of the fermentation supernatant was measured every 24 h. The results showed the derived peptide LfcinB-W4,10 had good antibacterial activity against Staphylococcus aureus, and the bacteriostatic ring diameter was the largest after 96 h induction. In conclusion, this study lays a foundation to further explore the relationship between function and structure of bovine lactoferrin peptides.

Cite this article

LYU Zili , ZHANG Enpeng , GUO Aizhen , LUO Bin , LIANG Xin , SHAN Xudong , ZHUANG Jing , ZHANG Xia , WANG Liang . Design of a recombinant bovine lactoferricin peptide and its expression in Pichia pastoris[J]. Food and Fermentation Industries, 2019 , 45(15) : 24 -29 . DOI: 10.13995/j.cnki.11-1802/ts.020507

References

[1] GONZ LEZ-CH VEZ S A, AR VALO-GALLEGOS S, RASC N-CRUZ Q. Lactoferrin: Structure, function and applications [J]. Int J Antimicrob Ag, 2009, 33(4): 1-8.
[2] PANTELEEV P V, BOLOSOV I A, BALANDIN S V, et al. Structure and biological functions of β-hairpin antimicrobial peptides [J]. Acta Naturae, 2015, 7(1): 37-47.
[3] SHESTAKOV A, JENSSEN H, NORDSTRM I, et al. Lactoferricin but not lactoferrin inhibit herpes simplex virus type 2 infection in mice [J]. Antiviral Res, 2012, 93(3): 340-345.
[4] BO L Y, LI T J, ZHAO X H. Effect of Cu/Mn-fortification on in vitro activities of the peptic hydrolysate of bovine lactoferrinagainst human gastric cancer BGC-823 cells[J]. Molecules, 2019, 24(7):1 195.
[5] AHMADINIA K, YAN D, ELLMAN M, et al. The anti-catabolic role of bovine lactoferricin in cartilage [J]. Biomolecular Concepts, 2013, 4(5): 495-500.
[6] STRM M B, REKDAL O, SVENDSEN J S. Antibacterial activity of 15-residue lactoferricin derivatives [J]. Chem Biol Drug Des, 2000, 56(5): 265-274.
[7] VEGA S C, MART NEZ D A, CHAL M D S, et al. Design, synthesis and evaluation of branched RRWQWR-based peptides as antibacterial agents against clinically relevant gram-positive and gram-negative pathogens[J]. Front Microbiol, 2018, 2(9):329.
[8] SINHA M, KAUSHIK S, KAUR P, et al. Antimicrobial lactoferrin peptides: The hidden players in the protective function of a multifunctional protein [J]. Int J Pept, 2013:1-12.
[9] PRITCHARD S R, KAILASAPATHY K. Dairy Ingredients for Food Processing [M]. Oxford, U K: Wiley-Blackwell, 2011:3-33.
[10] CHOU H T, KUO T Y, CHIANG J C, et al. Design and synthesis of cationic antimicrobial peptides with improved activity and selectivity against Vibrio spp. [J]. Int J Antimicrob Agents, 2008, 32(2): 130-138.
[11] EDWARDS I A, ELLIOTT A G, KAVANAGH A M, et al. Contribution of amphipathicity and hydrophobicity to the antimicrobial activity and cytotoxicity of β-hairpin peptides [J]. Acs Infectious Diseases, 2016, 2(6): 442-450.
[12] HOLLMANN A, MART-NEZ M, NOGUERA M E, et al. Role of amphipathicity and hydrophobicity in the balance between hemolysis and peptide-membrane interactions of three related antimicrobial peptides [J]. Colloid Surf. B:Biointerfaces, 2016, 141: 528-536.
[13] SUN C, LI Y, CAO S, et al. Antibacterial activity and mechanism of action of bovine lactoferricin derivatives with symmetrical amino acid sequences[J]. Int J Mol Sci, 2018, 19(10):76.
[14] DRAGO-SERRANO M E, CAMPOS-RODRIGUEZ R, CARRERO J C, et al. Lactoferrin and peptide-derivatives: Antimicrobial agents with potential use in nonspecific immunity modulation[J]. Curr Pharm Des, 2018,24(10):1 067-1 078.
[15] BRANDENBURG L O, MERRES J, ALBRECHT L J, et al. Antimicrobial peptides: Multifunctional drugs for different applications [J]. Polymers, 2012, 4(1): 539-560.
[16] HAMMAMI R, FLISS I. Current trends in antimicrobial agent research: Chemo- and bioinformatics approaches [J]. Drug Discov Today, 2010, 15(13-14): 540-546.
[17] WANG G, XIA L, ZHE W. APD3: The antimicrobial peptide database as a tool for research and education [J]. Nucleic Acids Res, 2016, 44(Database issue): D1 087-D1 093.
[18] BERTONI M, KIEFER F, BIASINI M, et al. Modeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology [J]. Sci Rep, 2017, 7(1): 10 480.
[19] HANIF W F, SHANKAR B R, PRATIMA G, et al. CAMPR3: A database on sequences, structures and signatures of antimicrobial peptides [J]. Nucleic Acids Res, 2016, 44(Database issue):D1 094-D1 097.
[20] CREGG J M. DNA-mediated transformation [J]. Methods Mol Biol, 2007, 389: 27-42.
[21] SEMPLE J I, LEHNER B. Single and dual drug selection for transgenes following bombardment of Caenorhabditis species[J]. Methods, 2014, 68(3):409-416.
[22] JAYANTA S, PLANTZ B A, MEHMET I, et al. Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: Case study with recombinant ovine interferon-tau [J]. Biotechnol. Bioeng, 2010, 89(1): 102-112.
[23] NI Z, ZHOU X, SUN X, et al. Decrease of hirudin degradation by deleting the KEX1 gene in recombinant Pichia pastoris [J]. Yeast, 2008, 25(1): 1-8.
Outlines

/