| Inclusion bodies were isolated by a modification of the method of Dekker et al. (22). Induced cultures were transferred to polypropylene bottles, chilled on ice, and harvested by centrifugation in a Sorvall GS3 rotor at 4 °C for 20 min at 4000 rpm (2700 × g). Cell pellets were thoroughly resuspended in 30 ml of ice-cold 50 mM Tris-HCl, 40 mM EDTA, pH 8.0 (per liter of culture) followed by the addition of 7.5 g of sucrose, 6 mg of lysozyme, and phenylmethylsulfonyl fluoride at 100 µg/ml. After incubation on ice for 30 min, the cells were lysed by osmotic shock by the addition of 30 ml of ice-cold 50 mM Tris-HCl, 40 mM EDTA, pH 8.0, and 100 µg/ml phenylmethylsulfonyl fluoride and incubated another 30 min. The lysates were transferred to 250 ml of polypropylene centrifuge bottles and chromosomal DNA sheared by repeated rounds (3-5) of sonication with a Branson Sonifier 250 equipped with a 1-cm diameter tip. Lysates were maintained on ice during each round consisting of several minutes of sonication (50% duty cycle, output 60) followed by several minutes to cool down. Triton X-100 (0.1% final concentration) was added to the sonicate prior to centrifugation in a Sorvall GSA rotor at 4 °C for 30 min at 5500 rpm (4500 × g). The large, pinkish-brown pellets were resuspended in an equal volume (60 ml) of ice-cold 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, 1 mM DTT (TED) by sonication, recentrifuged, and washed again with TED buffer. The twice-washed inclusion body pellets were resuspended in 2.0 ml of TED (per liter of initial culture) and transferred to Eppendorf tubes for storage at 20 °C. Protein concentration (approximately 18 mg/ml) was estimated by the method of Bradford (23) by solubilizing microliter volumes of a 10-fold dilution of resuspended inclusion bodies directly in the phosphoric acid/methanol reagent which was then diluted with water. HPLC-purified DPPC protein and bovine serum albumin gave similar responses in the dye-binding assay, indicating that bovine serum albumin standard curves can be used to estimate DPPC protein concentration, as has been shown previously to be feasible for DPP dimer from S2 cells and for the BMP family of proteins in general (13). Insufficient sonication resulted in loose, jelly-like pellets of inclusion bodies due to contamination with high molecular weight DNA which could be eliminated by additional cycles. As an alternative to sonication, a French press could also be employed and may be more practical with larger preparations.
In Vitro Folding-- Inclusion bodies suspended in TED buffer were centrifuged briefly (~2 min) in a microcentrifuge and solubilized at an estimated concentration of 2.5 mg/ml in buffered chaotrope. The final molarity of chaotrope is not significantly lowered by addition of the inclusion bodies, which were diluted more than 70-fold from an estimated initial concentration of 180 mg of protein/ml pellet. 8 M urea or 6 M GdmCl, buffered with 50 mM Tris-HCl, 2 mM EDTA, pH 8.0, 1 mM DTT, gave equivalent results. After incubation for 1-2 h at ambient temperature, insoluble debris was removed by a second brief centrifugation, and the solubilized protein was diluted 50-fold (final concentration 50 µg/ml), rapidly with stirring, into pre-chilled folding buffer (50 mM Tris-HCl, 2 mM EDTA, pH 8.0, 33 mM CHAPS detergent (1.8% w/v), 1.25 M NaCl, 2 mM reduced glutathione, and 1 mM oxidized glutathione) and incubated for at least 72 h at 4 or 15 °C. |