High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris
High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris
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DOI：
10.1016/j.pep.2004.09.006
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发表时间：
2005-02-01
影响因子：
1.6
通讯作者：
Kondo, H
中科院分区：
文献类型：
--
作者：
Li, L;Wang, JX;Kondo, H
对虾素（Penaeidins）是一种新的抗菌肽家族的成员，该家族的成员组成性地产生并储存在对虾的血细胞中，对细菌和真菌具有抗菌活性。本文将编码成熟Ch-penaeidin肽的DNA序列克隆到pPIC9K载体中，并转化为毕赤酵母。用增加G418浓度的方法筛选转化细胞的多拷贝质粒。通过表型和PCR鉴定携带Chp基因染色体整合的阳性菌落。转化后的细胞用甲醇诱导后，SDS-PAGE和Western blotting显示产生了类似于6100 Da的重组CHP （rCHP）表达产物。大规模表达表明，在最佳条件下，在最高产chp的pastoris克隆中，rCHP的产率为108 mg/L。液相分析表明，rCHP对部分革兰氏阴性菌和革兰氏阳性菌均有抑菌活性，但对部分真菌的抑菌活性较低。通过阳离子交换色谱纯化rCHP和随后的自动氨基酸测序发现，在n端存在4个额外的氨基酸（YVEF），属于裂解融合信号肽；这些残留物可能是抗真菌活性降低的原因。总之，这些观察结果表明，rCHP是一种有效的抗菌肽，可以在酵母中成功地高水平生产，因此可能是实际使用的潜在抗菌候选物。(C) 2004爱思唯尔公司版权所有。
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.