Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter.

Microbial Cell Factories
Yasuhiro FujinoKatsumi Doi

Abstract

Thermostable enzymes are commonly produced in mesophilic hosts for research and bioengineering purposes. However, these hosts do not overexpress the active forms of some biologically functional thermoenzymes. Therefore, an efficient thermophilic expression system is needed. Thermus thermophilus contains an easily manipulable genome and is therefore among the best candidate microbes for a "hot" expression system. We previously identified a strong and inducible promoter that was active in T. thermophilus under supersaturated silica conditions. Here, we report a new heterologous gene expression system based on a silica-inducible promoter in T. thermophilus. A Thermus sp. A4 gene encoding thermostable β-galactosidase was cloned as a reporter gene into the expression vector pSix1, which contains a selection marker that confers thermostable resistance to hygromycin and a 600 bp DNA region containing a putative silica-inducible promoter. β-galactosidase activity was 11-fold higher in the presence than in the absence of 10 mM silicic acid. SDS-PAGE revealed a prominent band corresponding to 73 kDa of β-galactosidase, and this enzyme was expressed as an active and soluble protein (yield: 27 mg/L) in Thermus but as an inclusion body in E...Continue Reading

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Mar 20, 2016·Applied and Environmental Microbiology·Yasuhiro FujinoKatsumi Doi

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Citations

Apr 21, 2021·Bioscience, Biotechnology, and Biochemistry·Takeshi Ikeda

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Datasets Mentioned

BETA
AP008226
LC428096
LC504201

Methods Mentioned

BETA
PCR
Protein Extraction
Protein Assay
size exclusion chromatography

Software Mentioned

Excel

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