Abstract
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.
Highlights
The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities
Often times, the gathering of reagents to preform a specific experiment can be more difficult than the execution of the experiment itself. This can especially true of experiments that examine the molecular interactions between proteins, which require the expression and purification of recombinant protein(s) from both eukaryotic and prokaryotic systems. Both systems have unique advantages for recombinant protein expression: prokaryotic E. coli allow for large quantities of recombinant protein to be and rapidly expressed and S. cerevisiae allow for eukaryotic proteins to be expressed with native binding partners and for in vivo function assessment
The vectors described here have several advantages; (1) genes can be cloned into the vectors in a ligation-independent method by homologous recombination in yeast, (2) a screen for protein expression and functionality can be used to identify positive clones in yeast before isolation of the plasmid DNA, (3) a protein can be expressed in both bacterial and yeast from the same vector, (4) a Glutathione Stransferase (GST)-fusion tag, which can be removed by the treatment with tobacco etching virus (TEV) protease, is provided to enhance protein solublility and purification, and (5) the entire protocol is very time efficient taking less than a week (5 days, 6 hours of hands-on time) to complete
Summary
The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. The vectors described here have several advantages; (1) genes can be cloned into the vectors in a ligation-independent method by homologous recombination in yeast, (2) a screen for protein expression and functionality can be used to identify positive clones in yeast before isolation of the plasmid DNA, (3) a protein can be expressed in both bacterial and yeast from the same vector, (4) a GST-fusion tag, which can be removed by the treatment with TEV protease, is provided to enhance protein solublility and purification, and (5) the entire protocol is very time efficient taking less than a week (5 days, 6 hours of hands-on time) to complete. These are the first examples of dual promoter vectors that allow for one-step cloning for both bacterial and yeast expression
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