Formin proteins: purification and measurement of effects on actin assembly.
We describe methods for expressing and isolating formin proteins from a wide range of species and comparing quantitatively their effects on actin assembly. We first developed these procedures for purification of S. cerevisiae formins Bni1 and Bnr1 but have extended them to mammalian formins, including mouse mDia1 and mDia2 and human Daam1. Thus, the approach we describe should be universally applicable to the purification and analysis of formins from any eukaryote. Formins expressed in yeast rather than bacteria usually have improved solubility, yield, and actin assembly activity. Yields are 200-500 microg purified formin per liter of yeast culture. For some applications bacterial expression and purification is preferable, and these methods are also described. For expression of most formins, in either yeast or bacteria, we recommend using an amino terminal 6xHis affinity tag. Active FH1-FH2 containing fragments of the formins Bni1, Bnr1, mDia1, mDia2, and Daam1 are all digomeric. However, they nucleate actin filaments with variable efficiencies, as high as one actin filament per formin complex. In the last section, we outline fluorometric methods for measuring and quantitatively analyzing the in vitro activities of formins on actin nucleation and processive capping of actin filaments.
Rosenstiel Center, Brandeis University, Waltham, MA, USA.
James B
JB
Moseley
Sankar
S
Maiti
Bruce L
BL
Goode
D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000302:isolation & purification* / Q000502:physiology*; D008840:Microfilament Proteins / Q000302:isolation & purification* / Q000502:physiology*; D011721:Pyrenes; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D011721:Pyrenes; C030984:pyrene