A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression

IF 1.2 Q2 Biochemistry, Genetics and Molecular Biology
J. Rodríguez-Mejía, Abigail Roldán-Salgado, J. Osuna, E. Merino, P. Gaytán
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引用次数: 5

Abstract

Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions.
绿色荧光蛋白基因开头密码子缺失增强蛋白表达
重组蛋白的表达是蛋白质工程和生物技术的关键问题之一。在评估蛋白质产生和结构功能研究的不同模型中,绿色荧光蛋白(GFP)是首选模型之一,因为它在细胞和分子研究中作为报告者的重要性。本研究分析了不同GFP蛋白氨基端附近密码子缺失对荧光的影响。我们的研究包括增强型GFP (EGFP)、红移型GFP和红移型EGFP中的Gly4缺失。在T7或Trc启动子的控制下,对Gly4缺失突变体及其对应的野生型突变体进行转录,并分析其表达模式。根据荧光基因版本的类型,观察到不同的荧光结果。对核糖体结合位点附近的RNA二级结构进行的计算机分析揭示了它们的最小自由能与GFP的产生之间的直接关系。对这些结果的综合分析,包括SDS-PAGE分析,使我们得出这样的结论:表达Gly4缺失的不同版本gfp的细胞的荧光改善是由于通过降低其mRNA前导区RNA二级结构的稳定性来增强核糖体结合位点的可及性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Molecular Microbiology and Biotechnology
Journal of Molecular Microbiology and Biotechnology 生物-生物工程与应用微生物
CiteScore
3.90
自引率
0.00%
发文量
0
审稿时长
>12 weeks
期刊介绍: We are entering a new and exciting era of microbiological study and application. Recent advances in the now established disciplines of genomics, proteomics and bioinformatics, together with extensive cooperation between academic and industrial concerns have brought about an integration of basic and applied microbiology as never before.
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