High-throughput expression in microplate format in Saccharomyces cerevisiae.
We have developed a high-throughput technology that allows parallel expression, purification, and analysis of large numbers of cloned cDNAs in the yeast Saccharomyces cerevisiae. The technology is based on a vector for intracellular protein expression under control of the inducible CUP1 promoter, where the gene products are fused to specific peptide sequences. These N-terminal and C-terminal epitope tags allow the immunological identification and purification of the gene products independent of the protein produced. By introducing the method of recombinational cloning we avoid time-consuming re-cloning steps and enable the easy switching between different expression vectors and host systems.
Holz
Caterina
C
Berlin Technical University, Institute for Biotechnology, Germany.
Lang
Christine
C
D002352:Carrier Proteins; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D008668:Metallothionein / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
C500769:CUP1-1 protein, S cerevisiae; D002352:Carrier Proteins; D011994:Recombinant Proteins; C007121:copper thionein; D008668:Metallothionein