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The nucleotide composition of the spacer sequence influences the expression yield of heterologously expressed genes in Bacillus subtilis

Paper ID Volume ID Publish Year Pages File Format Full-Text
23017 43409 2014 7 PDF Available
Title
The nucleotide composition of the spacer sequence influences the expression yield of heterologously expressed genes in Bacillus subtilis
Abstract

•A strong influence of the spacer sequence on the expression yield of GOIs is shown.•Changing the nucleotide composition of the spacer changes the expression level.•A polyadenylate-moiety as spacer increased the yield, but only moderately.•The effect of the spacer sequence is specific to the gene of interest.•The effects of the spacer might be exerted via the folding of the transcript.

Bacillus subtilis is a commonly used host for the heterologous expression of genes in academia and industry. Many factors are known to influence the expression yield in this organism e.g. the complementarity between the Shine–Dalgarno sequence (SD) and the 16S-rRNA or secondary structures in the translation initiation region of the transcript. In this study, we analysed the impact of the nucleotide composition between the SD sequence and the start codon (the spacer sequence) on the expression yield. We demonstrated that a polyadenylate-moiety spacer sequence moderately increases the expression level of laccase CotA from B. subtilis. By screening a library of artificially generated spacer variants, we identified clones with greatly increased expression levels of two model enzymes, the laccase CotA from B. subtilis (11 fold) and the metagenome derived protease H149 (30 fold). Furthermore, we demonstrated that the effect of the spacer sequence is specific to the gene of interest. These results prove the high impact of the spacer sequence on the expression yield in B. subtilis.

Keywords
Heterologous expression; Translation initiation; Spacer sequence; Folding of transcript; Bacillus subtilisGOI, gene of interest; P, probability (in significance testing); R, correlation coefficient; rRNA, ribosomal RNA; SD, Shine–Dalgarno; nt, nucleotide(
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The nucleotide composition of the spacer sequence influences the expression yield of heterologously expressed genes in Bacillus subtilis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 191, 10 December 2014, Pages 214–220
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us