Purification of Human β and γ-actin from Budding Yeast

Abstract

Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified -actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins and there is an underlying concern that these proteins perform differently with actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human {beta}- or {gamma}-actin (i.e., cytoplasmic actins), we developed Saccharomyces cerevisiae strains that express each as their sole source of actin. Both {beta}- or {gamma}-actin purified in this system polymerize and interact with various binding partners, including profilin, mDia1 (formin), fascin, and thymosin-{beta}4 (T{beta}4). Notably, T{beta}4 and profilin bind to {beta}- or {gamma}-actin with higher affinity than to -actin, emphasizing the value of testing actin ligands with specific actin isoforms. These reagents will make specific isoforms of actin more accessible for future studies of actin regulation.