DNase I affinity column chromatography

DNase I binds tightly to a globular actin (G-actin) monomer and forms a complex,
although the physiological function of this complex has not yet been elucidated. A DNase I
affinity column has been widely used to identify and isolate not only G-actin but also several
kinds of plant G-actin-binding-proteins, including a fragmin-like protein (Yamashiro et al.
2001), an actin-filament-severing protein (Fan et al. 2004), a gelsolin-like protein (Huang et al.
2004), and villins (Yokota et al. 2005), which are all adsorbed onto the column through aG-actin-DNase I interaction.
DNase I (grade II; Roche Diagnostics, Mannheim, Germany) was coupled to Affi-gel 10resin (Bio-Rad, Richmond, VA, USA) according to the manufacture’s instructions. Ten to 15ml Affi-gel 10 resin was rinsed with 250 ml cold deionized water, then equilibrated with 200ml Buffer A (10 mM CaCl2, 1 mM PMSF, and 100 mM HEPES-KOH at pH 7.5) on a glassfilter. Fifty mg DNase I was dissolved in 20 to 30 ml Buffer A and dialyzed in 500 ml Buffer
A at 4oC. A dialysate was mixed with the resin gently on a rotator at 4oC overnight, following
which 2-amonoethanol (pH 8.0) was added to the final concentration of 100 mM and further
mixed gently on a rotator at 4oC for 1 hour. After washing thoroughly with Buffer A, the resin
was packed into a column, such as a disposal syringe.
Plant materials, such as seedling of Arabidopsis (Fig. 1) and germinating pollen (Yokotaet al. 2005), were homogenized in Buffer B (60 mM KCl, 1 mM MgCl2, 0.2 mM ATP, 2 mM
DTT, 1 mM PMSF, 0.2 mg/ml leupeptin, and 30 mM HEPES-KOH at pH 7.5) supplemented
with 0.2 M sucrose, 0.2 mM CaCl2, and 1% casein, which potentially protects cytoskeletal
proteins from degradation caused by endogenous proteases. After centrifugation at 15,000 gfor 10 minutes, the supernatant was further centrifuged at 100,000 g for 30 minutes. After
adjusting pH of a resulting supernatant to 7.5 with KOH, the extract was applied to the DNase
I affinity column pre-equilibrated with Buffer B supplemented with 1 mM CaCl2. After thecolumn was thoroughly washed with the pre-equilibrated solution, the adsorbed materials wereeluted successively with Buffer B supplemented with 5 mM EGTA, and then with 50%
formamide or 3 M guanine hydrochloride.
Figure 1 shows an elution pattern from the DNase I affinity column of the extractprepared from Arabidopsis seedling. The molecular masses of standard proteins are indicatedon the left in kDa. A polypeptide with a molecular mass of about 123 kDa, which is mostprobably Arabidopsis villin, was eluted with 5 mM EGTA (indicated by an arrow in lane A).
G-actin was dissociated from DNase I by the elution with 50% formamide (lane B). Generally,
since the actin molecules are substantially denatured, they are no longer able to polymerize
into actin filaments. Nevertheless, there is only one report that showed that G-actin preparedfrom animal tissues by a DNase I affinity column exhibited significant polymerization activity(Schafer et al. 1998).

Fan et al. (2004) Plant Physiology 136: 3979-3989.
Huang et al. (2004) Journal of Biological Chemistry 279: 23364-23375.
Schafer et al. (1998) Cell Motility and the Cytoskeleton 39: 166-171.
Yamashiro et al. (2001) Journal of Biochemistry 130: 243-249.
Yokota et al. (2005) Plant and Cell Physiology 46: 1690-1703.