Abstract:
: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.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Moseley JB,Maiti S,Goode BLdoi
10.1016/S0076-6879(06)06016-2subject
Has Abstractpub_date
2006-01-01 00:00:00pages
215-34eissn
0076-6879issn
1557-7988pii
S0076-6879(06)06016-2journal_volume
406pub_type
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