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Expression enhancement of bubaline somatotropin in E. coli through gene modifications in the 5′-end coding region

Paper ID Volume ID Publish Year Pages File Format Full-Text
24819 43539 2008 6 PDF Available
Title
Expression enhancement of bubaline somatotropin in E. coli through gene modifications in the 5′-end coding region
Abstract

This study addresses the problem of poor expression of somatotropin (ST) gene in E. coli and describes expression enhancement through silent and non-silent gene modifications. A series of constructs with codon optimization, substitution, deletion or addition in the 5′-region of the sequence encoding bubaline ST (BbST) were prepared. In the native form, the BbST expression was barely discernible on SDS-gel of the total E. coli cellular proteins (TCP). Introduction of silent and non-silent mutations in +2 to +8 codons, however, raised the expression levels to varying extents. In some constructs, a single base variation, i.e., G → A or G → C led to a remarkable increase in BbST expression (up to 28% of the TCP) whereas in the case of G → T substitution the expression dropped to undetectable levels. Deletion of native GCC codon and addition of CAUCAC repeat thrice at +2 position enhanced the expression up to 48%, while insertion of NGG codons at the same position caused just a modest increase in expression. Differences in expression appeared as if related to the nature of early downstream codons (especially +2) and the stability of mRNA secondary structure although the levels of intracellular mRNA pools, as analyzed by real-time RT-PCR were quite similar. Overall, the study highlights the importance of 5′-end codon adaptations in solving the problems encountered in expressing the eukaryotic genes in E. coli.

Keywords
Somatotropin; mRNA secondary structure; Translation initiation; Gene expression; T7lac promoter; PCR mutagenesis
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Expression enhancement of bubaline somatotropin in E. coli through gene modifications in the 5′-end coding region
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 135, Issue 2, 1 June 2008, Pages 134–139
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