TY - JOUR T1 - A novel recombinant DNA system for high efficiency affinity purification of proteins in <em>Saccharomyces cerevisiae</em> JF - bioRxiv DO - 10.1101/025205 SP - 025205 AU - Brian H. Carrick AU - Lixuan Hao AU - Philip J. Smaldino AU - David R. Engelke Y1 - 2015/01/01 UR - http://biorxiv.org/content/early/2015/08/21/025205.abstract N2 - Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. The tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background in a single step. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent will allow isolation of rare or unstable protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or chromatin-associated assemblies. ER -