Conformational determinants necessary for secretion of Paecilomyces thermophila beta-1,4-xylosidase that lacks a signal peptide

文献类型： 外文期刊

第一作者： Yang, Yalin

作者： Yang, Yalin;Li, Juan;Yu, Qiang;Hou, Junxiu;Gao, Chenchen;Li, Dong;Liu, Yang;Ran, Chao;Zhou, Zhigang

作者机构：

关键词： Non-classical secretion;Secretory conformational determinants;Blades;Escherichia coli

期刊名称：AMB EXPRESS （ 影响因子：3.298； 五年影响因子：3.427 ）

ISSN： 2191-0855

年卷期： 2018 年 8 卷

页码：

收录情况： SCI

摘要： In this study, we investigated the secretion mechanism of the hyper-secretion signal peptide-lacking beta-xylosidase PtXyl43, a non-classically secreted protein, from the fungus Paecilomyces thermophila in Escherichia coli BL21( DE3). PtXyl43 secretion is a two-step process, and the second step is accompanied by cell periplasmic leakage, indicating that PtXyl43 secretion is the result of semi-specific secretion. Homology modeling of PtXyl43 suggested that PtXyl43 had a canonical GH43 family beta-xylosidase structure containing five blades. Seventeen blade deletions or circular mutants were designed to identify the conformational motif(s) involved in secretion. These mutants were expressed as recombinant, codon-optimized proteins in E. coli. Notably, only mutants containing blades 2-4 were effectively secreted. Blades 2-4 are necessary for secretion, but it appears that blade 1 or 5 must be present to maintain the structure of blades 2-4. Simultaneous deletion of blades 1 and 5 dramatically reduces excretion. The covalent and sequential linking of blades of 2, 3 and 4 are important for the excretion of mutants, as separate blades of 2 and 3 or 3 and 4 abolishes excretion. Fusion with PtXyl43 promotes the excretion of GFP from the periplasm to the extracellular milieu, which suggested that PtXyl43 had the potential to carry proteins. This study provides new insights into secretory mechanism of secretable signal peptide-lacking proteins in E. coli. To our knowledge, this is the first to definitively identify the conformational determinants for secretion of a signal peptide-lacking GH43 family beta-xylosidase. This finding also has application potential for the secretion of recombinant proteins.

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