| Denaturation of Protein. Lysozyme at 50 mg/mL was denatured and reduced in 100 mM Tris-HCl buffer (pH8.5) containing 8 M urea, 30 mM DTT, and 1 mM EDTA. The mixture was incubated at 37 C for 3 h under N2. After the mixture reached room temperature, DTT was removed by a HiTrap desalting column (5 mL, Amersham Biosciences). Prior to separation, the column was
equilibrated with 25 mL of 100 mM Tris-HCl buffer (pH 8.5) containing 8 M urea and 1 mM EDTA, which was also used as the elution solution for separation. The flow rate was about 1.0 mL/min. The complete reduction of disulfide bonds and removal of DTT were confirmed by thiol titration according to Ellmans method. The lysozyme concentrations were determined by UV
absorbance at 280 nm using extinction coefficients of 2.63 and 2.37 cm·mg/mL for native and denatured forms, respectively.20 All UV absorbance measurements were performed with a Shimadzu UV-3101PC spectrophotometer.
Adsorption of Denatured Lysozyme (D-Lys) on Mesoporous Materials. With stirring, mesoporous materials (2 mg/mL) were dispersed in D-Lys solutions of various initial concentrations in 100 mM Tris-HCl buffer (pH 8.5) containing 6 M urea and 1 mM EDTA. The amount adsorbed was calculated by subtracting the UV absorbance at 280 nm of the supernatant after centrifugation from that of D-Lys before adsorption.
Elution of Entrapped Protein. D-Lys (0.6 mg/mL) was loaded in 10 mg of mesoporous materials by stirringfor 2 h. The protein-entrapped solids were recovered by centrifugation. Elution tests were performed by adding 100 mM Tris-HCl buffer solution (pH 8.5) containing 2 M urea, 1 mM EDTA, and PEG (MW 2000) at
various concentrations to the recovered solids with stirring at room temperature. The volume of elution buffer added was determined by the adsorption capacity, giving a mixture of 1 mg of D-Lys per mL of solution. For example, 1.6 mL of elution solution was added to protein-containing E-PMO-A (164 mg of protein per g). At different time intervals, eluted proteins in solution were removed after centrifugation, and the same amounts of fresh buffer solutions were added again.
Protein Refolding. By the batch dilution method, refolding was initiated by a rapid 30-fold dilution of concentrated protein solution into refolding buffer (100 mM Tris-HCl, 1 mM EDTA, 2 M urea, 1.5 mM GSH, 0.3 mM GSSG, pH 8.5) at room temperature. Identical aliquots of the refolding sample were withdrawn and immediately assayed for enzymatic activity. In the case of PMO-assisted refolding, D-Lys (0.6 mg/mL) was
first loaded into E-PMO-A (20 mg), as described above. After adsorption for 2 h, protein-containing E-PMO-A (164 mg/g) was recovered by centrifugation. The refolding buffer (3.2 mL of 100 mM Tris-HCl, pH 8.5) containing 2 M urea, 1 mM EDTA, 1.5 mM GSH, 0.3 mM GSSG, and PEG at various concentrations (from 10 to 75 mg/mL) was then added. After the solution was stirred
for 20 h, the supernatant was recovered by centrifugation. The concentrations of recovered protein were determined by the Bio-Rad protein assay method. Native lysozyme was used as a protein standard.
Protein Activity Assay. The lysozyme activity was assayed at room temperature by monitoring the decrease in UV absorbance at 450 nm of a Micrococcus lysodeikticus cell suspension (0.20 mg/mL in 67 mM sodium phosphate buffer, pH 6.2). Renatured lysozyme solutions were first diluted with 100 mM Tris-HCl
buffer (pH 8.5) to the desired concentrations. A diluted protein sample (100 L) was then mixed with 3.0 mL of the cell suspension. A linear decrease in absorbance was observed, and the slope from 10 to 60 s was used to calculate the refolding yield by comparing to a native lysozyme solution of identical protein concentration. The activity of native lysozyme of
equal concentration was 100%. |