Troubleshooting Methods for Purification of MBP-Tagged Recombinant Proteins
| Problem | Possible cause | Solution |
|---|---|---|
| Increased back pressure | High viscosity of solutions. | Use lower flow rates and/or dilute the sample. |
| Insufficient cell disruption. | Increase the efficiency of the mechanical cell disruption, e.g., increase sonication time. (Keep the sample on ice during sonication to avoid frothing and overheating as this may denature the target protein. Over-sonication can also lead to co-purification of host proteins with the target protein). Increase dilution of the cell paste before mechanical lysis, or dilute after lysis to reduce viscosity. Decrease flow rate during sample loading. | |
| Freezing/thawing of the unclarified lysate has increased precipitation and aggregation. | Centrifuge or pass through a 0.22 or 0.45 µm filter before application on the column. | |
| Column has clogged | Top filter is clogged. | Change top filter. If using an MBPTrap column, replace the column. |
| Cell debris in the sample may have clogged the column. | Clean the column according to Appendix 3 (Characteristics of Dextrin Sepharose High Performance products). Centrifuge and/or filter the sample through a 0.22 µm or a 0.45 µm filter or otherwise optimize sample pretreatment before loading the next sample. | |
| No or weak binding | Protein found in the flowthrough. | Buffer/sample composition is not optimal; check the pH and composition of the sample and binding buffer. pH should in general be above pH 7. |
| Factors in the crude extract interfere with binding. | Include glucose in the growth medium to suppress amylase expression. | |
| MBP-tag is not present. | Use protease-deficient E. coli expression strains. Add protease inhibitors during cell lysis. | |
| MBP-tag is not accessible. | Fuse the MBP-tag with the other protein terminus. Use another linker. | |
| Protein has precipitated in the column due to high protein concentration. | Clean the column according to instructions in to Appendix 3 (Characteristics of Dextrin Sepharose High Performance products). In the following run decrease the amount of sample, or decrease protein concentration by eluting with a linear gradient instead of step-wise elution. Try detergents or change the NaCl concentration. If an MBPTrap HP 1 ml column has been used, change to the larger MBPTrap HP 5 ml. This will reduce the final concentration, provided that the same amount of sample is applied. For quick scale-up, connect two or more columns in series by screwing the end of one column into the top of the next. Note, however, that connecting columns in series will increase back pressure. | |
| Contaminating proteins | Contaminants are short forms of the tagged protein. | Use protease-deficient E. coli expression strains. Add protease inhibitors after cell lysis. Fuse the MBP-tag with the other protein terminus. Check for the presence of internal translation initiation starts (for C-terminal MBP-tag) or premature termination sites (for N-terminal MBP-tag). Use EDTA in the sample and buffers. Keep the sample cold. |
| Contaminants are covalently linked to the recombinant protein via disulfide bonds. | Add reducing agents to all buffers for cell lysis and purification. Note that the yield may decrease. | |
| Contaminants are non-covalently linked to the recombinant protein. | Increase ionic strength in all buffers for cell lysis and purification (up to 1 M NaCl) or add mild detergents, 0.1% Tween, 0.1% CHAPS). Be careful since the binding of MBP to dextrin may be affected by the addition of non-ionic detergents. | |
| Unwanted air bubble formation | Unclarified lysates may increase air bubble formation during purification. | Attaching a flow restrictor in the chromatography system can prevent this. If a flow restrictor is attached, it is important to change the pressure limit to adjust for the extra pressure from the flow restrictor. Do not exceed the pressure limit for the column on the ÄKTA system. |
| Air bubbles may form due to decreased air solubility when columns stored at 2 °C to 8 °C are used immediately at room temperature. | Let the columns adapt to room temperature for some minutes before using them. |
Materials
Sorry, an unexpected error has occurred
Network error: Failed to fetch
Sign In To Continue
To continue reading please sign in or create an account.
Don't Have An Account?
