Part:BBa_K4348057
SUMO-his
The SUMO protein is a small, very soluble protein that when fused with your protein of interest, will make the protein more soluble on average. It can easily be cleaved off later with a protease. This is a useful tool if your protein is aggregating or misfolding into inclusion bodies.
Introduction
The McGill iGEM team set out to develop a cholesterol lowering probiotic as a preventative for cardiovascular disease. Both endogenously synthesized cholesterol and dietary cholesterol end up in the gut, where they are absorbed and sent around the body. McGill iGEM’s project consists of developing a novel metabolic pathway to convert cholesterol, which is absorbed in the gut, into coprostanol, a molecule that cannot be absorbed and is thus excreted from the gut. This metabolic pathway is then packaged in a probiotic bacterium, where it makes its way to the gut where it prevents cholesterol absorption through its conversion into coprostanol.
This tag aids us in the purification of the first enzyme, ismA, in this metabolic pathway we wish to engineer.
Biology
Due to difficulties purifying our own recombinant proteins stemming from inclusion bodies, the SUMO-His tag presented an elegant potential solution. SUMO, or Small Ubiquitin-like Modifier, is a ~10 kD protein that is highly soluble in hydrophilic buffer compositions. As such, when fused to the end of a recombinant protein that tends to end up in the insoluble fraction (inclusion bodies), the SUMO protein is sometimes able to pull the average solubility of the protein up so that it ends up in the soluble fraction.
The SUMO-His tag also has a his-tag on its N-terminus, allowing you to identify your recombinant protein via western blot and purify it using affinity column chromatography. In addition, the SUMO-His tag may also be cleaved after purification by incubating with a SUMO-His protease that recognizes the tertiary structure of SUMO.
Results
Anti-his Western Blot of SUMO-ismA and ismA (no SUMO) protein purification. ismA-SUMO was detected in the elution of the ismA-SUMO sample group, indicating that it was successfully purified out from the inclusion bodies. However, the only detectable ismA in the ismA (no SUMO) sample was found in the cell lysate, indicating that all the ismA was stuck completely in the inclusion bodies.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 281
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 281
Illegal NheI site found at 64 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 281
Illegal BglII site found at 170
Illegal BamHI site found at 361 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 281
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 281
- 1000COMPATIBLE WITH RFC[1000]
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