Production of recombinant proteins requires the easy culturing, fast expression and high yields, however, one major problem is the insoluble expression in E.coli expression system. Maltose binding protein (MBP) tag is usually applied as an useful protein solubility partner to enhance the solubility of recombinant protein much more effective than GST fusion tag and TRx fusion tag.
MBP is anaturally occurring protein encoded by the malE gene, which is about 42 kDa in molecular weight. The major responsibilities of MBP are to uptake, breakdown and transport maltodextrin. MBP tag is mostly fused to the N-terminus to increase recombinant soluble expression. Creative Biostructure has developed combinatorial-tagging methods for custom needs. For example, using MBP tag as solubility-enhancing tag combined with a GST tag or His tag as the purification tag.
Advantages of MBP-tagged Proteins:
1. MBP-tagged proteins are usually able to achieve high yields in the cytoplasm of E. coli since the juxtaposition leads to efficient translation initiation.
2. MBP tag can be used to keep its passenger proteins from proteolytic degradation.
3. MBP tag is a natural affinity tag, which can be used to enhance purification of the passenger proteins.
4. MBP-tagged proteins are with remarkably enhanced solubility.
Disadvantages of MBP-tagged Proteins:
1. It is difficult to achieve target protein by cleavage because of the steric hindrance from the MBP tag.
2. The amylose resin, which is used to purify MBP-tagged proteins, is relatively expensive and fragile.
3. Target proteins sometimes precipitated after cleavage.
4. MBP-tagged proteins do not bind to the amylose resin efficiently in some cases.
MBP-tagged Protein Purification Troubleshooting
1. MBP-tagged protein is poorly soluble.
Not every MBP-tagged protein is highly soluble. You may try to reduce the culture temperature to 30°C or even after the addition of IPTG to increase the solubility. The improvement can be very dramatic in most cases. Using MBPs from different organisms is another possible approach.
2. MBP-tagged proteins do not bind or poorly bind to amylose resin.
There is about 20% of the constructed MBP-tagged proteins do not efficiently bind to amylose resin since the fusion protein aggregates and the passenger protein is improperly folded. You may try to increase the temperature from 4 °C to room temperature when perform amylose affinity chromatography. Constructing vectors that are designed to express MBP-tagged proteins with additional purification tags is another solution to solve this problem.
3. Precipitation of target protein occurs after MBP cleavage.
It is probably caused by improperly folding or aggregation preference of the target protein in its native state. You may vary the pH, metal ions, buffer composition, or other conditions without guarantee. Creative Biostructure advises you to double check the solubility of the passenger protein before purification of this target protein.
Creative Biostructure has devoted to MBP-tagged protein expression and purification for years. We are happy to accelerate your project by our resourceful and professional technical support platform. Please see the related services for more information and feel free to Contact Us.