Histidine-tagged wild-type yeast actin: its properties and use in an approach for obtaining yeast actin mutants

Wild-type and an N-terminal 6-histidine-tagged actin have each been expressed by using a yeast strain that contains the actin gene on a plasmid and not on the chromosome. Yeast strains have also been constructed that use two plasmids, one expressing the wild-type protein and the other the 6-histidine-tagged protein. Yeast cells can be grown with either plasmid alone or with both plasmids together and appear to be normal in that the growth rates of all the yeast strains are quite similar, as is the morphology of the yeast cells. The polymerization properties of the 6-histidine-tagged actin appear almost identical to wild-type actin expressed from the chromosome. When the wild-type and 6-histidine-tagged actin are coexpressed, they can be purified by standard techniques and then separated using nickel-nitrilotriacetate chromatography. The method can be used to prepare actin mutants including those that are nonfunctional or might not support yeast growth for other reasons.