This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wei, Q. Articles by Cha, H. J. Search for Related Content PubMed PubMed Citation Articles by Wei, Q. Articles by Cha, H. J. Agricola Articles by Wei, Q. Articles by Cha, H. J.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, September 2005, p. 5038-5043, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5038-5043.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Quande Wei,^1,4 Young Soo Kim,^3,4 Jeong Hyun Seo,^3,4 Woong Sik Jang,² In Hee Lee,² and Hyung Joon Cha^3,4*

Department of Pathogenic Biology, Sun Yat-sen University, Guangzhou 530080, People's Republic of China,¹ Department of Life Science, Hoseo University, Asan 336-795, Korea,² Department of Chemical Engineering,³ Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea⁴

Received 27 January 2005/ Accepted 3 April 2005

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 (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 (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.

---

* Corresponding author. Mailing address: Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea. Phone: 82-54-279-2280. Fax: 82-54-279-2699. E-mail: hjcha{at}postech.ac.kr.

---

Applied and Environmental Microbiology, September 2005, p. 5038-5043, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5038-5043.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Kim, Y. S., Cha, H. J. (2006). High-Throughput and Facile Assay of Antimicrobial Peptides Using pH-Controlled Fluorescence Resonance Energy Transfer.. Antimicrob. Agents Chemother. 50: 3330-3335 [Abstract] [Full Text]