| After washing, the inclusion bodies
were denatured in 10 ml of denaturing buffer (6 M
guanidinium chloride, 20 mM Tris?HCl, pH 7.4, 50
mM NaCl, 13 from stock 253 of the commercial cocktail of proteases inhibitors, 0.7 mg/ml pepstatin, and 20mM DTT). The high concentration of reducing agent
present in this buffer ensures that proteins are homogeneously reduced and unfolded. The denaturing solution was pipetted up and down to ensure complete solubilization of the inclusion bodies, followed by centrifugation for 15 min at 10000 rpm at 4°C in the Sorvall RC 5C-Plus centrifuge to separate the solubilized
denatured protein from cell debris. The cleared
supernatant was collected and the protein concentration determined by Bradford assay (Bio-Rad). If necessary more denaturing solution was added to achieve a final protein concentration of around 10?0 mg/ml. The solution of unfolded protein can be kept for a few
days at 4°C, and it was found best to leave the solution a few hours or overnight before use, to allow for complete denaturation.
Purification under denaturing conditions. As an
initial purification step, gel filtration using Superdex 200 prep grade 16/60 column (Pharmacia), was performed under denaturing conditions using FPLC (Pharmacia) controlled manually or through the program UNICORN. The column was equilibrated overnightwith denaturing buffer, plus 5 mM DTT and no protease inhibitors. Since the maximum sample capacity
for this size of column is 5 ml, it was necessary to split the sample over two or more runs and pool the fractions containing Tc1A. The column flow rate was 0.6 ml/min, and 1.8-ml fractions were collected from the second chromatographic peak that appeared after approximately 51.6 ml of flowthrough (approx. 1 h 30 min). Occasionally, the samples to be pooled together were checked by SDS?PAGE and stained with Coomassie
blue (R-250) (Sigma). The amount of sample
analyzed by SDS?PAGE was diluted four times in loading buffer (5 ml sample/20 ml loading buffer) to minimize the distortion of the bands due to the large amount of salt present in the samples. Fractions were selected according to their purity, avoiding those with low-molecular-weight contaminants. The pooled protein was adjusted with denaturing buffer to #2 mg/ml (50 ml final volume).
Refolding by gel filtration chromatography. Five
milliliters of denatured protein (2 mg/ml) was loaded on to a second Superdex 200 prep grade 16/60 column (Pharmacia), this time equilibrated with refolding buffer (10% glycerol, 25 mM phosphate buffer, pH 5.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM DTT) with a flow
rate of 0.6 ml/min. Fractions of 3 ml were collected. The chromatographic pattern of Tc1A refolding is always highly repetitive (Fig. 2). The peak that belongs to soluble and active protein appeared after approximately 72 ml (approx. 2 h). The purity of the fractions was checked by SDS?PAGE as described above. Multiple
loadings of 5 ml were necessary and the best
fractions from each run were pooled together (approx. 9?2 ml of sample collected per run). Total protein concentration was ;0.5 mg/ml, calculated by the Bradford
assay or by using UV, knowing that the e280 for
Tc1A is 76,200 (as calculated by the program PHD-sec (27)). The refolded protein at this stage shows a high degree of purity (around 95%), but there remain some contaminants of slightly lower molecular weight which are not resolved by gel filtration, so after refolding, an ion-exchange purification step was added. |