کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2022185 | 1069283 | 2006 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Rapid and easy thermodynamic optimization of the 5â²-end of mRNA dramatically increases the level of wild type protein expression in Escherichia coli
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
Low levels of expression in Escherichia coli are often observed when using wild type proteins. The addition of an N-terminal His-tag to these same proteins dramatically improves the level of expression. We therefore concluded that post-transcriptional regulation and in particular translational regulation are probably influenced by the presence of the tag. The RNAfold program was used to analyze the 5â²-end of the encoding mRNA, and more precisely the area encompassing the Shine-Dalgarno region and the initiation codon ATG. We observed that hairpin loops can be formed and that the stability of these loops correlates with the level of protein expression in E. coli. Our recently developed cloning technology by PCR fragment integration allows us to easily and rapidly introduce mutations anywhere within a gene. In our studies, we used this technology to destabilize the predicted hairpin by introducing silent mutations within the first 72 nucleotides of the coding sequence. As a result of the decreased stability of the RNA hairpins, we could significantly increase the level of expression of wild type proteins and without having to rely on the use of tags in E. coli. In addition, our studies allow us to predict whether or not a protein will be expressed without additional engineering of its encoding gene.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 45, Issue 2, February 2006, Pages 374-380
Journal: Protein Expression and Purification - Volume 45, Issue 2, February 2006, Pages 374-380
نویسندگان
Régis Cèbe, Martin Geiser,