Drag&Drop cloning in yeast.
We have developed a set of vectors that have enhanced capabilities for efficiently constructing and expressing differentially tagged fusion proteins using Drag&Drop cloning in the yeast Saccharomyces cerevisiae. The pGREG vectors are based on the pRS series with an additional general kanR selection marker. In vivo homologous recombination is used to introduce genes of interest into galactose-inducible expression vectors (pGREGs), permitting the formation of amino-terminal fusions. The vectors all contain common regions for recombination that flank the stuffer fragment. Introduction of common recombination sequences at the end of PCR fragments will permit the cloning of genes without the need for specific restriction sites. In this process, the selectable stuffer HIS3 gene is replaced by successful gene integration, and a screen for loss of the selection marker identifies potential recombinants. Due to the modular structure of the vectors, genes introduced into one vector can be readily transferred by in vivo recombination to all other members of the vector system, thus permitting rapid and easy Drag&Drop construction of a series of tagged proteins. The pGREG series combines features for expression, tagging, integration, localization and library construction with the advantage of obtaining immediate results from sub-sequent experiments. This Drag&Drop system also allows efficient cloning and expression of heterologous genes in large-scale experiments.
Jansen
Gregor
G
Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6. gregor.jansen@mcgill.ca
Wu
Cunle
C
Schade
Babette
B
Thomas
David Y
DY
Whiteway
Malcolm
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins