His-tag and GST-tag
His-tag and GST-tag protein fusions are commonly used for heterologous protein isolation from E.coli BL21. However, when expressing a eukaryotic protein in prokaryotic systems, you might experience protein degradation and protein precipitation.In this case, or if you plan on examining post translational protein modifications such as protein phosphorylation, you should consider a eukaryotic expression system. While this results in protein modifications that are closer to those of native proteins, they might give some problems when used in combination with some purification tags.
Check our Protein Purification Troubleshoot for tips on perfecting your experiment.
Protein size is also something to consider. The relative size of your tag of choice compared to the protein might interfere with protein folding. For example, it is not advised to use Glutathione S-transferase (GST) fusion for the isolation of small protein for this reason. An alternative would be to use immunoaffinity chromatography, which does not require tagged proteins and can be used for natural sources.
Explore our article: Malaria - Humanity's Deadliest Predator, for more information on the research uses of Protein Enrichment Kits.
The buffer used in extraction and purification might also interfere with the protein function or protein assays. High detergent concentration can interfere with protein folding which affects protein activity or protein-protein binding. This can be problematic when determining protein concentration, in particular using the Bradford method.
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Video: David Johnson, PhD/YouTube