A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid

BMC Biotechnology

Research Article

S Brookhart Shields¹  Charlotte A Williams²  Namita Sinah²  Robert C Piper² 

[1] Department of Biology, Luther College, 52101, Decorah, IA, USA;Department of Molecular Physiology and Biophysics, Roy J and Lucille A Carver College of Medicine, University of Iowa, 52242, Iowa City, IA, USA;

关键词: Recombinant Protein Expression;    URA3 Gene;    Tobacco Etching Virus Protease;    Target Plasmid;    Ampicillin Resistance Gene;   

DOI  :  10.1186/1472-6750-12-54

 received in 2012-07-05, accepted in 2012-08-17,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundThe ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both E. coli and S. cerevisiae.ResultsA set of expression vectors was made that can express the same open reading frame in E. coli (via the T7 phage promoter) and in S. cerevisiae (via the CUP1 or MET25 promoter). These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins.ConclusionsThe dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications.

【 授权许可】

Unknown   
© Sinah et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.