Dual cl 10111/13/2022 ![]() ![]() If you’re working with an uncharacterized protein, or a protein for which a good antibody has not been developed (and just because your protein has a commercially available antibody, that doesn’t mean it’s a good one), then your first step towards detecting, immunoprecipitating, or purifying that protein may be to fuse an affinity tag to it. Tags for affinity and purificationĪn affinity tag, generally a relatively small sequence of amino acids, is basically a molecular leash for your protein. MBP tags can help with solubility issues, but scientists may also choose to add smaller proteins, such as Thioredoxin A (TrxA) that improve disulfide bond formation in order to help keep your protein soluble. Overexpression can also lead to insolubility, and aggregated protein is not useful protein. Small ubiquitin-related modifier (SUMO) can help with folding and stabilization, as can maltose-binding protein (MBP). You can get your bacteria to produce massive amounts of protein, but if it’s not folded correctly, there’s no point in crystallizing it or testing its function. Prokaryotes can also have a hard time folding eukaryotic proteins. Though there are a number of chemical and peptide-based proteosome inhibitors, glutathione S-transferase (GST), which can be fused to recombinant proteins for one-step purification with glutathione, can also protect against proteolysis. Eukaryotes and some bacteria deploy proteosomes to degrade what the cell might consider junk protein. Energy and cellular resources are being spent to make something the cell doesn’t need to make. What are some of the hurdles to overcome in order to overexpress a recombinant protein? It is not generally in a cell’s best interest to overexpress a protein. After several decades of trying to address these challenges, researchers have amassed a considerable molecular tool box of tags and fusion proteins to aid in the expression and purification of recombinant proteins. Biochemists and molecular biologists who need to overexpress and purify proteins can face any number of technical challenges depending on their protein of interest. Thus far Plasmids 101 has covered GFP and its related fluorescent proteins, which are sometimes used as tags for detection however, those are just one (admittedly large) class of common fusion protein tags. As depicted in the accompanying cartoon, they have a multitude of uses including (but not limited to) purification, detection, solubilization, localization, or protease protection. Protein tags are usually smallish peptides incorporated into a translated protein. ![]()
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