High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

doi:10.1016/j.pep.2004.09.006

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	High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris
	Li, L; Wang, JX; Zhao, XF ; Kang, CH ; Liu, N ; Xiang, JH; Li, FH ; Sueda, S ; Kondo, H
	2005-02-01
发表期刊	PROTEIN EXPRESSION AND PURIFICATION
ISSN	1046-5928
卷号	39 期号:2 页码:144-151
文章类型	Article
摘要	Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a similar to6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use. (C) 2004 Elsevier Inc. All rights reserved.; Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a similar to6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use. (C) 2004 Elsevier Inc. All rights reserved.
关键词	Chinese Shrimp (Fenneropenaeus Chinensis) Antimicrobial Peptide Pichia Pastoris Recombinant Expression Penaeidin
学科领域	Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
DOI	10.1016/j.pep.2004.09.006
URL	查看原文
收录类别	SCI
语种	英语
WOS记录号	WOS:000226437000003
引用统计	被引频次：82[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/3102
专题	实验海洋生物学重点实验室
作者单位	1.Shandong Univ, Sch Life Sci, Jinan 250100, Peoples R China 2.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China 3.Kyushu Inst Technol, Dept Biochem Engn & Sci, Iizuka, Fukuoka 8208502, Japan
推荐引用方式 GB/T 7714	Li, L,Wang, JX,Zhao, XF,et al. High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris[J]. PROTEIN EXPRESSION AND PURIFICATION,2005,39(2):144-151.
APA	Li, L.,Wang, JX.,Zhao, XF.,Kang, CH.,Liu, N.,...&Kondo, H.(2005).High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris.PROTEIN EXPRESSION AND PURIFICATION,39(2),144-151.
MLA	Li, L,et al."High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris".PROTEIN EXPRESSION AND PURIFICATION 39.2(2005):144-151.

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