Secretion and activity of antimicrobial peptide cecropin D expressed in Pichia pastoris

Guo,Chunhe; Huang,Yumao; Zheng,Hongyu; Tang,Liyun; He,Jun; Xiang,Linsheng; Liu,Dehui; Jiang,Houquan

doi:10.3892/etm.2012.719

Secretion and activity of antimicrobial peptide cecropin D expressed in Pichia pastoris

Authors:
- Chunhe Guo
- Yumao Huang
- Hongyu Zheng
- Liyun Tang
- Jun He
- Linsheng Xiang
- Dehui Liu
- Houquan Jiang
View Affiliations

Affiliations: College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong 510642, P.R. China
Published online on: September 24, 2012 https://doi.org/10.3892/etm.2012.719
Pages: 1063-1068

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

To express the antimicrobial peptide cecropin D in Pichia pastoris and determine the activity of the expressed product, four oligonucleotide fragments were synthesized in accordance with the available cecropin D sequences and a codon bias suitable for Pichia pastoris. Sequence fragments were phosphorylated, annealed, linked and cloned into the expression vector pGAPZαA and the yeast α‑mating factor signal peptide was used as the signal sequence. The P. pastoris SMD1168 cells were transformed by electroporation using the constructed recombinant plasmid pGAPZαA‑cecropin D. We were able to demonstrate by PCR that the cecropin D sequence had integrated into the P. pastoris genome. The expressed and secreted product was identified using Tricine‑SDS‑PAGE. Antibacterial activity was demonstrated using an agarose diffusion test and turbidimetry. The molecular mass of the recombinant cecropin D was estimated to be 3,900 Da. The recombinant cecropin D exhibited antibacterial activity for both Gram‑positive and Gram‑negative bacteria, suggesting that cecropin D was successfully expressed in P. pastoris. This approach holds great promise for antibacterial drug development.

View Figures

View References

1	Lee DG, Park JH, Shin SY, Lee SG, Lee MK, Kim KL and Hahm KS: Design of novel analogue peptides with potent fungicidal but low hemolytic activity based on the cecropin A-melittin hybrid structure. Biochem Mol Biol Int. 43:489–498. 1997.PubMed/NCBI
2	Steiner H, Hultmark D, Engström A, Bennich H and Boman HG: Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature. 292:246–248. 1981. View Article : Google Scholar
3	Hultmark D, Steiner H, Rasmuson T and Boman HG: Insect immunity: purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem. 106:7–16. 1980. View Article : Google Scholar : PubMed/NCBI
4	Hultmark D, Engström A, Bennich H, Kapur R and Boman HG: Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae. Eur J Biochem. 127:207–217. 1982. View Article : Google Scholar : PubMed/NCBI
5	Dickinson L, Russell V and Dunn PE: A family of bacteria-regulated, cecropin D-like peptides from Manduca sexta. J Biol Chem. 263:19424–19429. 1988.PubMed/NCBI
6	Morishima I, Suginaka S, Ueno T and Hirano H: Isolation and structure of cecropins, inducible antibacterial peptides, from silkworm Bombyx mori. Comp Biochem Physiol B. 95:551–554. 1990.PubMed/NCBI
7	Kylsten P, Samakovlis C and Hultmark D: The cecropin locus in Drosophila; a compact gene cluster involved in the response to infection. EMBO J. 9:217–224. 1990.PubMed/NCBI
8	Okada M and Natori S: Primary structure of sacrotoxin I, an antibacterial protein induced in the hemolymph of Sarcophaga peregrine (flesh fly) larvae. J Biol Chem. 260:7174–7177. 1985.PubMed/NCBI
9	Kobayashi S and Uchimiya H: Expression and integration of a foreign gene in orange (Citrus sinensis Osb.) protoplasts by direct DNA transfer. Jpn J Genet. 64:91–97. 1989. View Article : Google Scholar
10	Cregg JM, Cereghino JL, Shi J and Higgins DR: Recombinant protein expression in Pichia pastoris. Mol Biotechnol. 16:23–52. 2000. View Article : Google Scholar : PubMed/NCBI
11	Daly R and Hearn MT: Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit. 18:119–138. 2005.PubMed/NCBI
12	Damasceno LM, Pla I, Chang HJ, Cohen L, Ritter G, Old LJ and Batt CA: An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris. Protein Expr Purif. 37:18–26. 2004. View Article : Google Scholar : PubMed/NCBI
13	Minagawa S, Hikima J, Hirono I, Aoki T and Mori H: Expression of Japanese flounder c-type lysozyme cDNA in insect cells. Dev Comp Immunol. 25:439–445. 2001. View Article : Google Scholar : PubMed/NCBI
14	Lehrer RI, Rosenman M, Harwig SS, Jackson R and Eisenhauer P: Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol Methods. 137:167–173. 1991. View Article : Google Scholar : PubMed/NCBI
15	Jin FL, Xu X, Zhang W and Gu D: Expression and characterization of a housefly cecropin gene in the methylotrophic yeast, Pichia pastoris. Protein Expr Purif. 49:39–46. 2006. View Article : Google Scholar : PubMed/NCBI
16	Cipáková I, Hostinová E, Ganperik J and Velebný V: High-level expression and purification of a recombinant hBD-1 fused to LMM protein in Escherichia coli. Protein Expr Purif. 37:207–212. 2004.PubMed/NCBI
17	Valore EV and Ganz T: Laboratory production of antimicrobial peptides in native conformation. Methods Mol Biol. 78:115–131. 1997.PubMed/NCBI
18	Mølhøj M, Ulvskov P and Dal Degan F: Characterization of a functional soluble form of a Brassica napus membrane-anchored endo-1,4-beta-glucanase heterologously expressed in Pichia pastoris. Plant Physiology. 127:674–684. 2001.
19	Peres MdFS, Silva VC, Valentini SR and Gattás EAdL: Recombinant expression of glycerol-3-phosphate dehydrogenase using the Pichia pastoris system. J Mol Catal B Enzym. 65:128–132. 2010. View Article : Google Scholar
20	Johnson SC, Yang M and Murthy PP: Heterologous expression and functional characterization of a plant alkaline phytase in Pichia pastoris. Protein Expr Purif. 74:196–203. 2010. View Article : Google Scholar : PubMed/NCBI
21	Wu M and Hancock RE: Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane. J Biol Chem. 274:29–35. 1999. View Article : Google Scholar : PubMed/NCBI
22	Liang YL, Wang JX, Zhao XF, Du XJ and Xue JF: Molecular cloning and characterization of cecropin from the housefly (Musca domestica), and its expression in Escherichia coli. Dev Comp Immunol. 30:249–257. 2006. View Article : Google Scholar : PubMed/NCBI
23	Cabral KM, Almeida MS, Valente AP, Almeida FC and Kurtenbach E: Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inefficiency of the STE13 protease. Protein Expr Purif. 31:115–122. 2003.PubMed/NCBI
24	Skosyrev VS, Kulesskiy EA, Yakhnin LV, Temirov YV and Vinokurov LM: Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells. Protein Expr Purif. 28:350–356. 2003. View Article : Google Scholar : PubMed/NCBI
25	Aly R, Granot D, Mahler-Slasky Y, Halpern N, Nir D and Galun E: Saccharomyces cerevisiae cells harboring the gene encoding Sarcotoxin IA secrete a peptide that is toxic to plant pathogenic bacteria. Protein Expr Purif. 16:120–124. 1999. View Article : Google Scholar
26	Yamada K, Nakajima Y and Natori S: Production of reombinant sarcotoxin IA in Bombyx mori cells. Biochem J. 272:633–636. 1990.PubMed/NCBI
27	Cereghino JL and Cregg JM: Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 24:45–66. 2000. View Article : Google Scholar