Controlled expression of recombinant genes and preparation of cell-free extracts in yeast.
Biochemistry is an important experimental tool in the study of protein functions. Biochemical studies frequently involve overexpression of a cloned gene and purification of the recombinant protein. The yeast Saccharomyces cerevisiae provides an effective system for expression and purification of recombinant proteins owing to the ease of applying molecular techniques and obtaining large quantities of cells with a low cost. Additionally, complex biochemical processes such as transcription and DNA repair can be studied in yeast cell-free extracts in vitro, which benefit greatly from a large collection of well-defined mutant strains. Controlled gene expression and preparation of cell-free extracts are important techniques in the yeast system. Two commonly used inducible gene expression systems, the GAL1 promoter and the CUP1 promoter, are described. Protocols of preparing yeast whole cell extracts and nuclear extracts are presented, each of which is designed for specific applications.
Wang
Zhigang
Z
Graduate Center for Toxicology, University of Kentucky, Lexington, USA.
D002458:Cell Fractionation / Q000379:methods; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005822:Genetic Vectors; D009172:Mycology / Q000379:methods; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D011994:Recombinant Proteins