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


RESULTS
A 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.


CONCLUSIONS
The 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.
Department of Molecular Physiology and Biophysics, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Namita
N
Sinah
Charlotte A
CA
Williams
Robert C
RC
Piper
S Brookhart
SB
Shields
D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010450:Endopeptidases; C099488:TEV protease