Recombinant antimicrobial peptide hPAB-ß expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus.
Appl Microbiol Biotechnol
; 89(2): 281-91, 2011 Jan.
Article
em En
| MEDLINE
| ID: mdl-20857289
ABSTRACT
As a potential therapeutic agent, antimicrobial peptide has received increased attention in recent years. However, high-level expression of a small peptide with antimicrobial activity is still a challenging task. In this study, the coding sequence of antimicrobial peptide hPAB-ß, a variant derived from human beta-defensin 2, was cloned into pPIC9K vector and transformed into Pichia pastoris. P. pastoris transformants harbored with multi-copy plasmids were screened by G418 selection. When the transformed cells were induced by methanol, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed recombinant hPAB-ß products consisting of three protein species of 4,680.4, 4,485.3, and 4,881.9 Da at proportions of 58%, 36%, and 6%, respectively, which may be due to the incomplete processing of the fusion signal peptide of α-factor by the STE13 protease. Expressed hPAB-ß was secreted into the culture medium at a level of 241.2 ± 29.5 mg/L. Purified hPAB-ß with 95% homogeneity was obtained by 10 kDa membrane filtration followed by cation ion-exchange chromatography with a SP-Sepharose XL column. The two major protein species separated through a SOURCE 30RPC reverse phase chromatography column showed definite antimicrobial activities against Staphylococcus aureus. All 22 methicillin-resistant S. aureus (MRSA) isolates with multidrug resistance phenotype were sensitive to the recombinant hPAB-ß with minimal inhibitory concentrations of 8-64 µg/ml. Our results show that the methylotrophic yeast-inducible system is suitable for high-level expression of active hPAB-ß, and that expressed hPAB-ß in P. pastoris may be a potential antimicrobial agent against MRSA infection.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pichia
/
Expressão Gênica
/
Beta-Defensinas
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Staphylococcus aureus Resistente à Meticilina
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Antibacterianos
Limite:
Humans
Idioma:
En
Revista:
Appl Microbiol Biotechnol
Ano de publicação:
2011
Tipo de documento:
Article