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Microorganisms 2019, 7(1), 9; https://doi.org/10.3390/microorganisms7010009

An Alternative Platform for Protein Expression Using an Innate Whole Expression Module from Metagenomic DNA

Department of Biological Sciences and Research Center of Ecomimetics, College of Natural Sciences, Chonnam National University, Yongbong-ro, Buk-gu, Gwangju 61186, Korea
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Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Received: 8 November 2018 / Revised: 20 December 2018 / Accepted: 3 January 2019 / Published: 8 January 2019
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Abstract

Many integrated gene clusters beyond a single genetic element are commonly trapped as the result of promoter traps in (meta)genomic DNA libraries. Generally, a single element, which is mainly the promoter, is deduced from the resulting gene clusters and employed to construct a new expression vector. However, expression patterns of target proteins under the incorporated promoter are often inconsistent with those shown in clones harboring plasmids with gene clusters. These results suggest that the integrated set of gene clusters with diverse cis- and trans-acting elements is evolutionarily tuned as a complete set for gene expression, and is an expression module with all the components for the expression of a nested open reading frame (ORF). This possibility is further supported by truncation and/or serial deletion analysis of this module in which the expression of the nested ORF is highly fluctuated or reduced frequently, despite being supported by plentiful cis-acting elements in the spanning regions around the ORF such as the promoter, ribosome binding site (RBS), terminator, and 3′-/5′-UTRs for gene expression. Here, we examined whether an innate module with a naturally overexpressed gene could be considered as a scaffold for an expression system. For a proof-of-principle study, we mined a complete expression module with an innately overexpressed ORF in E. coli from a metagenomics DNA library, and incorporated it into a vector that had no regulatory element for expressing the insert. We obtained successful expression of several inserts such as MBP, GFPuv, β-glucosidase, and esterase using this simple construct without tuning and codon optimization of the target insert. View Full-Text
Keywords: protein expression; innate module; evolutionary optimization; expression vector; Escherichia coli protein expression; innate module; evolutionary optimization; expression vector; Escherichia coli
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Cheong, D.-E.; Park, S.-Y.; Lim, H.-D.; Kim, G.-J. An Alternative Platform for Protein Expression Using an Innate Whole Expression Module from Metagenomic DNA. Microorganisms 2019, 7, 9.

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