Last modified 3/27/01

Purification of Recombinant UNC-44 Fragments

The host strain BLR(DE3)pLysS (F^- ompT, hsdS_B(r_B^-m_B^-), gal, dcm, delta(srl-recA)306::Tn10(DE3)pLysS) (Novagen, Madison, WI) was used for expression of all recombinant proteins. BLR cultures containing the desired plasmid were diluted 1:100 in a liter of LB media (Sambrook et al., 1989) supplemented with ampicillin (150 ug/ml), tetracycline (10 ug/ml), and chloramphenicol (34 ug/ml), then induced with 0.1 mM IPTG, and grown overnight.

For the purification of AO271 recombinant protein, cells were harvested, 2.7 g of cells resuspended in 20 ml of Lysis Buffer (50 mM Tris pH. 8.0), freeze thawed (The BLR strain produces T7 lysozyme.), and sonicated. DNA was degraded by adding MgCl₂ to 10 mM and 10 ug of DNAse I, and the sample incubated on ice for 10 min or until viscosity decreased. Fifty ml of Detergent Buffer (0.2 M NaCl, 1% (w/v) deoxycholate, 1% NP-40, 20 mM Tris pH 7.5) were added and inclusion bodies pelleted by centrifugation at 5000 x g for 5-10 min at 4C. The pellet was resuspended in 2 ml of 0.5% Triton X-100 and 10 ug of DNAse I added if the pellet was gelatinous. Washes of the pellet were repeated as necessary to remove bacterial debris from the whitish inclusion bodies. The pellet was resuspended in 2 ml Denaturing Binding Buffer (pH 7.8) [8M urea, 0.02M sodium phosphate, 0.05M sodium chloride, pH 7.8] and adsorbed batchwise on a ProBond nickel chelating column according to the manufacturer’s directions (Invitrogen). The column was washed batchwise with 4 ml of Denaturing Binding Buffer (pH 5.3), then with 10 ml of Denaturing Binding Buffer (pH 4.0), and eluted with 10 ml of Denaturing Binding Buffer (pH 7.8) + 200 mM imidazole. The sample was dialyzed against PBS (Sambrook et al., 1989). The overall yield was 0.6-0.8 mg of AO271 protein.

The AO280 protein was prepared from a liter culture of bacteria. Cells (7.6 g) were resuspended and lysed, and the inclusion body preparation done as described above. The inclusion bodies were resuspended in Denaturing Binding Buffer, bound to a nickel column, the column washed as described by the manufacturer, and recombinant protein eluted with 200 mM imidazole in Denaturing Binding Buffer pH 7.8. The protein was dialyzed against Denaturing Binding Buffer pH 7.8, reloaded on a nickel-chelating column, and eluted with a 0-200 mM gradient of imidazole in Denaturing buffer pH 7.8.

Cells (5.6 g) were suspended in 20 ml of Lysis Buffer. An inclusion body preparation was done and the AO346 protein was found in the supernatants. Supernatants were combined and adsorbed batchwise to a nickel-chelating column. The column was washed batchwise with Denaturing Binding Buffer at pH 5.3, then at pH 4.0, and eluted with 10 ml Denaturing Binding Buffer (pH 7.8) + 50 mM imidazole. The sample was dialyzed against Denaturing Binding Buffer (pH 7.8), loaded on a nickel-chelating column, and eluted with a 20 ml 0-200 mM imidazole gradient in Denaturing Binding Buffer (pH 7.8). The protein was dialyzed against PBS. The overall yield was 0.8-1.0 mg of AO346 protein.

A liter culture was grown without induction overnight at 37C, then induced with 0.1 mM IPTG, and grown 5 hr at room temperature to reduce toxic effects of producing a hydrophobic protein. Cells (7.2 g) were resuspended in 50 ml of Guanidinium Lysis Buffer (Invitrogen), sonicated, centrifuged at 3000 x g for   15 min at 4C, and the supernatant loaded by gravity flow onto a 2 ml nickel chelating column. The column was washed batchwise with Denaturing Binding Buffer at pH 5.3, and then at pH 4.0. The column was eluted with 10 ml Denaturing Binding Buffer (pH 4.0) + 50 mM imidazole. The sample was dialyzed against Denaturing Binding Buffer (pH 7.8), loaded on nickel chelating column, and eluted with a 20 ml 0-100 mM imidazole gradient in Denaturing Binding Buffer (pH 7.8). The protein was dialyzed against PBS. The dialyzed protein gradually precipitated from the solution over a one or two day period. Overall yield was 75-100 ug of AO289 protein which was about 50% pure.

Both the AO263 and AO327 proteins appear in the supernatants of inclusion body preparations. The supernatants from inclusion body preparations of AO263 and AO327 proteins in Native Binding Buffer (0.02M sodium phosphate, 0.5M sodium chloride, pH 7.8) were separately bound to nickel columns. For AO263, the column was washed with Denaturing Binding Buffer (pH 6.0), and the AO263 protein was eluted with a similar buffer at pH 4.0. For AO327, the column was washed with Native Binding Buffer (pH 4.0), and final nickel column elution was done with Denaturing Binding Buffer containing 200mM imidazole. After dialysis against Native Binding Buffer, the protein was further purified on a T7-Tag antibody agarose column following the manufacturer’s instructions (Novagen). Relevant fractions were determined by western blotting with T7-Tag antibody (Novagen). The proteins were about 10% pure.