Overexpression of multisubunit replication factors in yeast.
Facile genetic and biochemical manipulation coupled with rapid cell growth and low cost of growth media has established the yeast Saccharomyces cerevisiae as a versatile workhorse. This article describes the use of yeast expression systems for the overproduction of complex multipolypeptide replication factors. The regulated overexpression of these factors in yeast provides for a readily accessible and inexpensive source of these factors in large quantities. The methodology is illustrated with the five-subunit replication factor C. Whole-cell extracts are prepared by blending yeast cells with glass beads or frozen yeast with dry ice. Procedures are described that maximize the yield of these factors while minimizing proteolytic degradation.
Burgers
P M
PM
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA.
D002455:Cell Division; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D010450:Endopeptidases / Q000378:metabolism; D005656:Fungal Proteins / Q000191:economics / Q000302:isolation & purification*; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D018398:Homeodomain Proteins; D015778:Minor Histocompatibility Antigens; D010957:Plasmids; D011480:Protease Inhibitors / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2; D051818:Replication Protein C; D012097:Repressor Proteins; D012440:Saccharomyces / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D011480:Protease Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; D051818:Replication Protein C