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Antimicrobial Agents and Chemotherapy, September 2009, p. 3683-3689, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00251-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Novel Expression Vector for Secretion of Cecropin AD in Bacillus subtilis with Enhanced Antimicrobial Activity{triangledown}

Xiang Chen,{dagger} Faming Zhu,{dagger} Yunhe Cao, and Shiyan Qiao*

National Key Laboratory of Animal Nutrition, National Feed Engineering Technology Research Center, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, People's Republic of China

Received 21 February 2009/ Returned for modification 17 April 2009/ Accepted 10 June 2009

Cecropin AD, a chimeric antimicrobial peptide obtained from cecropins, is effective at killing specific microorganisms. However, a highly efficient expression system is still needed to allow for commercial application of cecropin AD. For the exogenous expression of cecropin AD, we fused the cecropin AD gene with a small ubiquitin-like modifier (SUMO) gene and a signal peptide of SacB, while a Bacillus subtilis expression system was constructed based on Bacillus subtilis cells genetically modified by the introduction of an operon including an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible Spac promoter, a signal peptide of amyQ, and the SUMO protease gene. The recombinant cecropin AD was expressed, and 30.6 mg of pure recombinant protein was purified from 1 liter of culture supernatant. The purified cecropin AD displayed antimicrobial activity against some pathogens, such as Staphylococcus aureus and Escherichia coli, and was especially effective toward Staphylococcus aureus, with MICs of <0.05 µM (0.2 µg/ml). Stability analysis results showed that the activity of cecropin AD was not influenced by temperatures as high as 55°C for 20 min; however, temperatures above 85°C (for 20 min) decreased the antimicrobial activity of cecropin AD. Varying the pH from 4.0 to 9.0 did not appear to affect the activity of cecropin AD, but some loss of potency was observed at pH values lower than pH 4.0. Under the challenge of several proteases (proteinase K, trypsin, and pepsin), cecropin AD maintained functional activity. The results indicated that the recombinant product expressed by the designed Bacillus subtilis expression system was a potent antimicrobial agent and could be applied to control infectious diseases of farm animals or even humans.

* Corresponding author. Mailing address: National Key Laboratory of Animal Nutrition, National Feed Engineering Technology Research Center, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, People's Republic of China. Phone: 86-10-6273-1456. Fax: 86-10-6273-3688. E-mail: qiaoshy{at}mafic.ac.cn

{triangledown} Published ahead of print on 22 June 2009.

{dagger} Xiang Chen and Faming Zhu contributed equally to this work.

Antimicrobial Agents and Chemotherapy, September 2009, p. 3683-3689, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00251-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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