Engineering Pichia pastoris for Efficient Production of a Novel Bifunctional Strongylocentrotus purpuratus Invertebrate-Type Lysozyme

doi:10.1007/s12010-018-2753-z

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	Engineering Pichia pastoris for Efficient Production of a Novel Bifunctional Strongylocentrotus purpuratus Invertebrate-Type Lysozyme
	Huang, Peng 1; Shi, Jinlei2 ; Sun, Qingwen 3; Dong, Xianping 1; Zhang, Ning 4
	2018-10
发表期刊	APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY (IF:3.1[JCR-2023],2.9[5-Year])
ISSN	0273-2289
卷号	186 期号:2 页码:459-475
发表状态	已发表
DOI	10.1007/s12010-018-2753-z
摘要	Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5mg/L and purity of >99%. Recombinant sp-iLys reached its peak lytic activity of 8560U/mg at pH 6.0 and 30 degrees C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37 degrees C with the presence of 0.05M Na+. In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.
关键词	Invertebrate-type lysozyme Strongylocentrotus purpuratus Pichia pastoris Antimicrobial activity Isopeptidase activity
收录类别	SCI ; SCIE ; EI
语种	英语
资助项目	Natural Science Foundation of Shanghai[15ZR1421800]
WOS研究方向	Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS类目	Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS记录号	WOS:000443820700013
出版者	HUMANA PRESS INC
WOS关键词	SINGLE-STEP PURIFICATION ; TAPES-JAPONICA LYSOZYME ; I-TYPE LYSOZYMES ; EGG-WHITE ; SEA-URCHIN ; ANTIBACTERIAL ANALYSIS ; HETEROLOGOUS PROTEINS ; RECOMBINANT PROTEIN ; MOLECULAR-CLONING ; ASTERIAS-RUBENS
原始文献类型	Article
引用统计	正在获取...
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/27768
专题	生命科学与技术学院_公共教学平台_生物科学与生物技术基础实验教学中心
通讯作者	Dong, Xianping; Zhang, Ning
作者单位	1.Shanghai Univ Med & Hlth Sci, Sch Clin Med, Shanghai, Peoples R China 2.ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai, Peoples R China 3.Fudan Univ, Sch Life Sci, Shanghai, Peoples R China 4.Dalhousie Univ, Dept Physiol & Biophys, Halifax, NS, Canada
推荐引用方式 GB/T 7714	Huang, Peng,Shi, Jinlei,Sun, Qingwen,et al. Engineering Pichia pastoris for Efficient Production of a Novel Bifunctional Strongylocentrotus purpuratus Invertebrate-Type Lysozyme[J]. APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY,2018,186(2):459-475.
APA	Huang, Peng,Shi, Jinlei,Sun, Qingwen,Dong, Xianping,&Zhang, Ning.(2018).Engineering Pichia pastoris for Efficient Production of a Novel Bifunctional Strongylocentrotus purpuratus Invertebrate-Type Lysozyme.APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY,186(2),459-475.
MLA	Huang, Peng,et al."Engineering Pichia pastoris for Efficient Production of a Novel Bifunctional Strongylocentrotus purpuratus Invertebrate-Type Lysozyme".APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 186.2(2018):459-475.