3.3.7. Purification by Ion-Exchange Chromatography
1. Pack 25 mL Q-Sepharose into the glass column (~5-cm bed height). Equilibrate the column at flow rate of 2 mL/min with equilibration buffer. Use the AKTA purifier for ionexchange chromatography.
2. Filter the refolded protein through a 0.45-^m filter, and load onto the pre-equilibrated Q-Sepharose ion-exchange column at a flow rate of 2 mL/min. Wash the column using three-column volumes of equilibration buffer containing 10 mM NaCl to remove nonspecific contaminants.
3. Elute the bound r-hGH using a linear continuous gradient from 10 to 500 mM NaCl in equilibration buffer.
4. Homogeneous r-hGH in the form of monomer elute at concentrations of between 150 and 250 mM NaCl, whereas protein aggregates elute at higher ionic strength (see Note 11).
5. The samples containing homogeneous r-hGH bands on SDS-PAGE (Fig. 3) should be pooled (approx 20 mL) and dialyzed against a lower urea gradient in each dialysis change, changing after every 8 h as described in the following buffers.
a. First change: 250 mL 50 mM Tris-HCl, 1 mM EDTA, 1 M urea, 5% (w/v) sucrose, and 1 mM PMSF, pH 8.5.
b. Second change: 250 mL 50 mM Tris-HCl, 0.5 mM EDTA, 0.5 M urea, and 2.5% (w/v) sucrose, pH 8.5.
c. Third change: 250 mL 10 mM Tris-HCl, 0.5 mM EDTA, 0.25 M urea, and 1% (w/v) sucrose, pH 8.5.
d. Fourth change: 250 mL 10 mM Tris-HCl, and 1% (w/v) sucrose, pH 8.5.
6. When the dialysis step is complete, lyophilize the r-hGH and use for the subsequent gel filtration step (see Note 12).
3.3.2. Purification by Gel Filtration
1. Pack the gel filtration column with Sephacryl S-200 resin up to 96 cm. Use the liquid chromatography system for gel filtration.
2. Dissolve the lyophilized hGH (approx 14 mg) in 3-4 mL 10 mM Tris-HCl buffer containing 1% (w/v) sucrose. Filter the solution through a 0.45-^m filter to remove any aggregates (see Note 13).
3. Load the protein solution in the form of a layer on top of the gel filtration matrix.
4. Run the column at a flow rate of 20 mL/h using a peristaltic pump, and collect fractions.
5. Check each fraction by SDS-PAGE for the presence of hGH, which will elute after approximately half a column volume of buffer.
6. Pool all the protein fractions containing pure hGH, and dialyze against 10 mM Tris-HCl buffer containing 1% sucrose. Check by SDS-PAGE for purity.
7. Lyophilize the protein and store for physicochemical and biological assay. Determine the protein concentration using the Micro-BCA assay procedure (see Notes 14 and 15).
1. The postinduction time required after IPTG addition for maximum gene expression is different for varying proteins and depends on the host vector relationship. Most often, optimal induction time is 3-6-hours postinduction for inclusion body accumulation. Do not grow E. coli cells overnight after induction, as cell lysis may expose the inclusion bodies to the culture medium and create problems during purification. Use freshly grown, induced cells for the isolation of inclusion bodies and subsequent protein refolding.
2. Remove the dense inclusion body layer very carefully without disturbing the other layers. If the homogeneity of the inclusion bodies is not sufficient, repeat the ultracentrifugation sucrose gradient again using the pellet of the first ultracentrifugation step. Depending on the density of the inclusion bodies, they will be deposited at different places in the sucrose gradient after ultracentrifugation.
3. Do not freeze the purified inclusion bodies to be used at a later date for solubilization. It is always advisable to isolate, purify, and refold the protein into a soluble form without freezing the protein. For quantification of the amount of protein in inclusion bodies, completely solubilize them in detergent solution (1% SDS in the case of r-hGH), and undertake a protein estimation using the detergent-compatible protein assay kit as described. Add detergent to the protein standard solution to minimize error. The presence of EDTA interferes with the Micro-BCA protein assay.
4. There is some loss of r-hGH in the supernatant during deoxycholate treatment. However, this step helps in eliminating contaminating membrane proteins. Depending on the nature of the therapeutic protein, deoxycholate concentrations may need to be optimized. Some proteins will be solubilized in 1% deoxycholate solution, whereas for others, this will not be the case. Additional use of 0.5-1 M NaCl and/or 2 M urea sometimes helps in solubi-lization of contaminating cellular proteins and may help improve the purity of inclusion bodies.
5. Use gloves while performing SDS-PAGE (acrylamide is a neurotoxin). Do not pipet the pellet at the bottom of the microfuge tube during sample loading for SDS-PAGE. Rinse the syringe a few times with distilled water after loading each individual well. If E. coli cells or the inclusion body pellet does not dissolve in sample dye, use 10-20 |lL 10% SDS solution to dissolve them, then add SDS sample buffer for processing before loading onto the SDS-PAGE gel.
6. To determine the best buffer for solubilization of inclusion bodies of a particular protein, it is necessary to know the dominant forces that cause protein aggregation that lead to inclusion body formation. This can be determined by solubilization of aliquots of pure inclusion bodies in different buffers and monitoring the percent solubility either by protein assay or reduction in solution turbidity (16). Alternatively, a sparse matrix approach can be used to design the solubilization protocol for a particular protein (26). If disulfide bonds have an important role in the protein structure and stability, use P-mercaptoethanol in the solubilization buffer. If the protein of interest degrades at pH 12.5, adjust the pH of the buffer to pH 12.
7. Never use frozen inclusion body pellets for solubilization or keep the leftover pellet for solubilization at a later date. It is advisable to undertake the inclusion body purification, solubilization, and refolding in one attempt without storing the protein between any step.
8. Do not keep the solubilized protein at a high pH for any longer than absolutely necessary. Thiolate ion formation may result, leading to protein amidation and ultimately resulting in poor-quality bioactive protein. Use freshly prepared buffers, particularly adding the urea to the buffer just before the experiments to reduce cyanate ion formation. Carry out refolding at a low temperature to reduce the extent of protein aggregation during refolding.
9. The use of glycerol, sucrose, and 2 M urea helps to prevent protein aggregation during refolding and, more importantly, during gel filtration and lyophilization of the refolded protein. It is advisable to add these excipients to improve the protein stability during the different stages of processing.
10. Check for turbidity during the refolding process; otherwise, carry out the pulsatile-refolding process in large volumes, or dilute the solubilized protein to a concentration of 1 mg/mL before refolding. This helps to lower the aggregation of proteins. A different refolding process may be used instead of pulse dilution if this approach does not give a good recovery of soluble protein (24).
11. The recombinant protein aggregates will usually elute at a higher ionic strength than the monomer during ion-exchange chromatography. However, sometimes the aggregates will coelute with the monomer. If significant amounts of protein coelute with the monomer, collect the mixture, dialyze, and run the gel filtration step as described to recover the monomeric protein.
12. Carry out dialysis with decreasing concentrations of urea at each step. The risk of protein aggregation is high during the removal of urea. Depending on the nature of a protein, the dialysis time and stepwise decreases of urea can be optimized to reduce the extent of any protein aggregation.
13. Refolded protein should be soluble in aqueous buffer. If the lyophilized r-hGH does not dissolve in aqueous buffer, this indicates that the protein is not refolded correctly. Try different excipients or concentrations of sucrose during lyophilization to achieve a better recovery. Filter the sample, and only use the soluble protein for further purification by gel filtration.
14. Calculate the total amount of r-hGH recovered at the end of the gel filtration process. The recovery should be around 40-50% (considering that the starting amount of r-hGH was 50 mg). The total recovery after gel filtration should be approx 20 mg of pure hGH.
15. If the recovery of protein is very low using the gel filtration process, it may be necessary to use different methods of refolding (25) as described in previous chapters of this book. Refolding by column chromatography (27), reverse micelles (28), or microfiltration (29) can be used to improve the recovery of protein.
The authors thank Dr. Sandip K. Basu (Director, National Institute of Immunology,
New Delhi, India) for providing core facilities for recombinant protein research.
The work is also supported by grants from the Department of Biotechnology, Government of India to AKP and LCG.
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