Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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Purification of Bacterially Expressed Proteins from Inclusion Bodies |
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| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley | ||||||||||||||||||||||||
| Overview |
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| This protocol describes the procedure to purify insoluble recombinant proteins from the inclusion bodies in bacterial cells. | |||||||||||||||||||||||||
| Procedure |
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A. Optimal Conditions for Expression of the Highest Level of the Protein 1. Grow cultures to mid-log phase (Optical Density (OD) of 0.5 at a wavelength of 600nm) at the appropriate temperature and induce expression of the recombinant protein with the addition of IPTG to a final concentration of at least 1 mM. 2. Collect cells at various time points to determine the moment when protein expression is maximal (see Hint #2 and Protocol ID#455). B. Preparation of Inclusion Bodies 1. Grow an appropriate amount of cells (liters) for the optimal amount of time and repeat induction with IPTG. 2. Centrifuge the cells in a Sorvall® GS3 rotor at 7,000 X rpm (8000 X g) for 7 min at 4°C. Decant the supernatant. 3. Resuspend the cell pellet in 15 ml of ice-cold Buffer A per 500 ml culture. 4. Add 100 μl of 10 mg/ml Lysozyme per ml of resuspended cells (see Hint #3). 5. Incubate on ice for 15 min. 6. Sonicate each 15 ml cell suspension eight times for 5 seconds (with incubations on ice between sonications) with a Branson sonicator fitted with a microtip at setting #2. 7. Centrifuge the lysate at 90,000 X g for 30 min at 4°C. 8. Remove the supernatant. C. Isolation and Purification of Inclusion Bodies 1. Resuspend the cell pellet in 15 ml of Buffer A containing the appropriate amount of urea that will not lyse the inclusion bodies, but will solubilize cell debris and contaminating proteins (see Hint #4). 2. Sonicate for 5 seconds to break up pellet. 3. Use a syringe and 18 Gauge needle to further break up the pellet by drawing and extruding the suspension several times. Do not expect to homogenize the suspension. 4. Rotate the Urea-solubilized suspension at 4°C for 30 min. 5. Centrifuge the suspension at 90,000 X g for 30 min at 4°C. Discard the supernatant. 6. Resuspend the inclusion body pellet in Buffer A containing the amount of urea that will solubilize the recombinant protein in the inclusion bodies (see Hint #5). 7. Rotate the urea-solubilized protein at 4°C for 30 min. 8. Spin the suspension at 90,000 X g for 30 min at 4°C. COLLECT the supernatant. D. Protein Refolding 1. Dialyze the solubilized recombinant protein in sequentially decreasing concentrations of Urea for at least 2 to 2.5 hours per concentration in Buffer B. If the concentration of Urea used in Buffer B was 6 M, start dialyzing with 6 M, then 4 M, then 2 M, then 1 M, then 0.5 M, then no Urea in Buffer B (see Hint #6). Maintain a minimum of a 50:1 ratio of dialysis buffer to sample. |
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| Solutions |
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| BioReagents and Chemicals |
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DTT IPTG Glycerol PMSF EDTA Lysozyme Urea Tris Sodium Chloride |
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| Protocol Hints |
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. Typical strains of bacteria that may be employed as host are TG2 and the BL21(DE3) bacterial cell lines (which contain IPTG-inducible expression of T7 RNA polymerase). Strains that are deficient in cellular proteases are also suitable, as this may allow expression of unstable or short-lived proteins that might otherwise be poorly expressed. This protocol was developed using BL21(DE3)::pLysE as the host strain and pRSETA expression vectors. 3. If the host strain is BL21(DE3)::pLys, several cycles of freezing the cell suspension followed by rapid thawing will achieve sufficient cell lysis. This should be followed by sonication. This process can be attempted for other strains, but the degree of cell lysis will vary from strain to strain and should be empirically determined. 4. Empirically determine on a pilot scale the optimal concentration of Urea in Buffer A. This generally will be between 1 M and 6 M final concentration of Urea. Adding NaCl to a final molarity of 0.5 to 1 M may improve the solubility of contaminating proteins. 50 ml conical tubes may be easier to handle than manipulations in Ti70 tubes. 5. Repeat empirical determination as in Hint #4 with the consideration that the concentration of Urea will be higher than the concentration chosen from Hint #4. Six molar Guanidine HCl can also be used if the recombinant protein is difficult to solubilize. 6. Each step down in Urea needs to be watched carefully to determine whether or not the protein precipitates. If the protein precipitates as the concentration of Urea decreases, adding NaCl to 500 mM (or more) and decreasing the protein concentration to 100 μg/ml (or less) may improve the solubility. |
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