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Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli.
- Source :
-
Applied and environmental microbiology [Appl Environ Microbiol] 2005 Sep; Vol. 71 (9), pp. 5038-43. - Publication Year :
- 2005
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Abstract
- Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein ( approximately 33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP ( approximately 2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.
- Subjects :
- Amino Acid Sequence
Antimicrobial Cationic Peptides chemistry
Antimicrobial Cationic Peptides genetics
Base Sequence
Escherichia coli drug effects
Escherichia coli metabolism
Gene Expression Regulation, Bacterial
Microbial Sensitivity Tests
Molecular Sequence Data
Occlusion Body Matrix Proteins
Peptides chemistry
Peptides genetics
Peptides pharmacology
Protein Engineering methods
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins isolation & purification
Recombinant Proteins pharmacology
Staphylococcus aureus drug effects
Viral Proteins genetics
Viral Structural Proteins
Antimicrobial Cationic Peptides metabolism
Escherichia coli genetics
Peptides metabolism
Recombinant Fusion Proteins metabolism
Viral Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0099-2240
- Volume :
- 71
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- Applied and environmental microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 16151084
- Full Text :
- https://doi.org/10.1128/AEM.71.9.5038-5043.2005