Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli.
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.
Wei
Quande
Q
Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.
Kim
Young Soo
YS
Seo
Jeong Hyun
JH
Jang
Woong Sik
WS
Lee
In Hee
IH
Cha
Hyung Joon
HJ
D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D000077183:Occlusion Body Matrix Proteins; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000187:drug effects; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins
D023181:Antimicrobial Cationic Peptides; D000077183:Occlusion Body Matrix Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C460842:halocidin; C009829:polyhedrin protein, Nucleopolyhedrovirus