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Rational design of translational pausing without altering the amino acid sequence dramatically promotes soluble protein expression: A strategic demonstration

Paper ID Volume ID Publish Year Pages File Format Full-Text
23058 43415 2014 10 PDF Available
Title
Rational design of translational pausing without altering the amino acid sequence dramatically promotes soluble protein expression: A strategic demonstration
Abstract

•A novel method to improve heterogeneous protein production by translational pausing-guided design of synonymous mutations.•2000x increase of Cyanovirin-N soluble expression in E. coli w/o altering the amino acid sequence or culturing condition.•3'-terminal translational pausing is effective for protein co-translational folding.•This strategy introduces new possibilities for the production of bioactive recombinant proteins.

The production of many pharmaceutical and industrial proteins in prokaryotic hosts is hindered by the insolubility of industrial expression products resulting from misfolding. Even with a correct primary sequence, an improper translation elongation rate in a heterologous expression system is an important cause of misfolding. In silico analysis revealed that most of the endogenous Escherichia coli genes display translational pausing sites that promote correct folding, and almost 1/5 genes have pausing sites at the 3′-termini of their coding sequence. Therefore, we established a novel strategy to efficiently promote the expression of soluble and active proteins without altering the amino acid sequence or expression conditions. This strategy uses the rational design of translational pausing based on structural information solely through synonymous substitutions, i.e. no change on the amino acids sequence. We demonstrated this strategy on a promising antiviral candidate, Cyanovirin-N (CVN), which could not be efficiently expressed in any previously reported system. By introducing silent mutations, we increased the soluble expression level in E. coli by 2000-fold without altering the CVN protein sequence, and the specific activity was slightly higher for the optimized CVN than for the wild-type variant. This strategy introduces new possibilities for the production of bioactive recombinant proteins

Keywords
Protein folding; Translation pausing; Synonymous substitutions; Cyanovirin-N; Antiviral activity; Escherichia coli
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Rational design of translational pausing without altering the amino acid sequence dramatically promotes soluble protein expression: A strategic demonstration
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 189, 10 November 2014, Pages 104–113
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