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Engineering the Salt-Inducible Ectoine Promoter Region of Halomonas elongata for Protein Expression in a Unique Stabilizing Environment.

Engineering the Salt-Inducible Ectoine Promoter Region of Halomonas elongata for Protein Expression in a Unique Stabilizing Environment.
Author Information (click to view)

Stiller LM, Galinski EA, Witt EMHJ,


Stiller LM, Galinski EA, Witt EMHJ, (click to view)

Stiller LM, Galinski EA, Witt EMHJ,

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Genes 2018 03 289(4) pii 10.3390/genes9040184

Abstract

It has been firmly established that organic osmolytes (compatible solutes) of halophilicandhave positive effects on conformation and activity of proteins, and may therefore improve their functional production. In particular, the amino acid derivative ectoine is known for its conformational stabilization, aggregation suppression, and radical protection properties. The natural producer and industrial production strainaccumulates ectoine in the cytoplasm, and as a result offers a unique stabilizing environment for recombinant proteins. For the construction of broad hoast range vector systems with fluorescent reporter proteins, we chose the salt-inducible promoter region of the ectoine gene cluster (). A closer inspection of the genetic background revealed that its combination of sigma 38 (σ) and sigma 70 (σ) promoters was followed by a weak ribosomal binding site (RBS). This inspired a systematic approach for the construction of a-based vector series with a synthetic RBS region using the RBS Calculator v2.0, which resulted in a greatly improved salt-dependent expression-even in a deletion construct lacking the σpromoter. To expand the application range of this expression system, we looked further into the possible export of recombinant proteins into the periplasm. Bothandleader sequences fromproved to be suitable for directed periplasmic transport into an extreme environment of freely selectable ionic strength.

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