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 express ed by using a yeast strain that contains the actin gene on a plasmid and no t on the chromosome. Yeast strains have also been constructed that use two plasmids, one expressing the wild type protein and the other the 6-histidin e-tagged protein. Yeast cells can be grown with either plasmid alone or wit h both plasmids together and appear to be normal in that the growth rates o f all the yeast strains are quite similar, as is the morphology of the yeas t cells. The polymerization properties of the 6-histidine-tagged actin appe ar almost identical to wild-type actin expressed from the chromosome. When the wild-type and 6-histidine-tagged actin are coexpressed, they can be pur ified by standard techniques and then separated using nickel-nitrilotriacet ate chromatography. The method can be used to prepare actin mutants includi ng those that are nonfunctional or might not support yeast growth for other reasons.