| Isolation and purification of recombinant DNase-I and scFv-DNase-I proteins
Cells were harvested and lysed as previously described (Deonarain and Epenetos, [1998]; Deonarain et al., [1997]), resuspending the bacteria in a buffer containing 50 mM Tris-HCl (pH 7.8), 2 mM CaCl2, 1 mM MgCl2. The soluble fraction was used to purify the soluble DNase-I by IMAC on nickel-Sepharose. The column was equilibrated with 50 mM Tris-HCl (pH 7.8), 300 mM NaCl (buffer C). The filtered protein was applied and the column washed once with buffer (C), followed by a wash with the same buffer at pH 6.0 and a further wash with the same buffer containing 10 mM imidazole, pH 6.0. Elutions were performed with the above buffer at pH 6.0 containing increasing imidazole concentrations (50 mM to 1 M). Fractions were collected and analysed by SDS-PAGE and Western blotting. Those containing the purest protein (100 mM imidazole elutions) were pooled and concentrated through 10 kDa m.w. cut-off membrane units and dialysed overnight at 4°C in PBS. Final purification was achieved by gel filtration (below).
Insoluble material was treated and purified under denaturing IMAC conditions as described by Deonarain and Epenetos ([1998]): the column was equilibrated in 8 M urea, 0.1 M Tris-HCl (pH 8.0), 300 mM NaCl (buffer D). Binding and washes were performed as described above, but the buffers used were buffer D (pH 8.0), buffer D (pH 6.0) and, for elutions, buffer D (pH 6.0) containing increasing concentrations of imidazole. Fractions were pooled as above and concentrated through a 10 kDa cut-off membrane unit to about 5 mg/ml. The protein was reduced by addition of solid dithioeritol (DTE) to a final concentration of 0.3 M for 2 hr at room temperature.
The purified, denatured and reduced DNase-I sample was rapidly diluted 100-fold into 10 mM Tris-HCl (pH 7.6), 10 mM CaCl2, 1 mM MgCl2 at room temperature and incubated for about 1 hr. The protein was then concentrated through a 10 kDa cut-off membrane unit, dialysed against the same buffer and analysed for activity. The scFv-DNase-I chimera was rapidly diluted 100-fold into a modified buffer of 0.1 M Tris-HCl (pH 8.0), 0.5 M L-arginine, 4 mM GSSG (oxidised), 10 mM CaCl2 and 1 mM MgCl2 (pre-chilled to 10°C) and incubated at 10°C for 48 to 72 hr. The sample was then concentrated through a 30 kDa cut-off membrane, dialysed overnight against PBS and analysed. The protein sample was then filter-sterilised through a 0.2 m filter and subsequently stored in the above buffer at 4°C. |