Expression of BmSPI38 tandem multimers in <i>Escherichia coli</i> and its antifungal activity.

Youshan LI; Yuan Wang; Rui Zhu; Xi Yang; Meng Wei; Zhaofeng Zhang; Changqing Chen

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Expression of BmSPI38 tandem multimers in Escherichia coli and its antifungal activity.

Author: Youshan LI ¹ ; Yuan WANG ¹ ; Rui ZHU ² ; Xi YANG ³ ; Meng WEI ¹ ; Zhaofeng ZHANG ¹ ; Changqing CHEN ⁴
Author Information

1. College of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong 723001, Shaanxi, China.
2. Qinba Mountain Area Collaborative Innovation Center of Bioresources Comprehensive Development, Hanzhong 723001, Shaanxi, China.
3. Qinba State Key Laboratory of Biological Resources and Ecological Environment (Incubation), Shaanxi University of Technology, Hanzhong 723001, Shaanxi, China.
4. Shaanxi Province Key Laboratory of Bio-resources, Hanzhong 723001, Shaanxi, China.
Publication Type:Journal Article
Keywords: Bombyx mori; antifungal activity; heterologous expression; protease inhibitor; structural homogeneity; tandem multimer
MeSH: Animals; Antifungal Agents/pharmacology*; Escherichia coli/metabolism*; Proteins/metabolism*; Protease Inhibitors/chemistry*; Bombyx/chemistry*; Saccharomyces cerevisiae/metabolism*; Peptide Hydrolases
From: Chinese Journal of Biotechnology 2023;39(10):4275-4294
CountryChina
Language:Chinese
Abstract: The aim of this study was to prepare tandem multimeric proteins of BmSPI38, a silkworm protease inhibitor, with better structural homogeneity, higher activity and stronger antifungal ability by protein engineering. The tandem multimeric proteins of BmSPI38 were prepared by prokaryotic expression technology. The effects of tandem multimerization on the structural homogeneity, inhibitory activity and antifungal ability of BmSPI38 were explored by in-gel activity staining of protease inhibitor, protease inhibition assays and fungal growth inhibition experiments. Activity staining showed that the tandem expression based on the peptide flexible linker greatly improved the structural homogeneity of BmSPI38 protein. Protease inhibition experiments showed that the tandem trimerization and tetramerization based on the linker improved the inhibitory ability of BmSPI38 to microbial proteases. Conidial germination assays showed that His₆-SPI38L-tetramer had stronger inhibition on conidial germination of Beauveria bassiana than that of His₆-SPI38-monomer. Fungal growth inhibition assay showed that the inhibitory ability of BmSPI38 against Saccharomyces cerevisiae and Candida albicans could be enhanced by tandem multimerization. The present study successfully achieved the heterologous active expression of the silkworm protease inhibitor BmSPI38 in Escherichia coli, and confirmed that the structural homogeneity and antifungal ability of BmSPI38 could be enhanced by tandem multimerization. This study provides important theoretical basis and new strategies for cultivating antifungal transgenic silkworm. Moreover, it may promote the exogenous production of BmSPI38 and its application in the medical field.