Selecting Fusion Proteins for Prokaryotic Expression
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Selection of a suitable fusion partner to enhance soluble expression and serve as an affinity tag for recombinant protein production in prokaryotic expression systems (E. coli) is a crucial step that should not be overlooked during construct design.
Both Maltose Binding Protein (MBP) and Glutathione S-Transferase (GST) often improve yields of soluble protein at reasonable costs. Amylose resin is used for purification of MBP, while glutathione resin is utilized for GST.
Both fusion partners have limitations such as incompatibility with reducing and denaturing agents and low binding affinity for the amylose resin, so an alternative tag for purification method may need to be considered. For instance, GST and MBP may be paired with a His-tag to drive high level expression by the fusion partner and provide an alternative purification handle via the His-tag.
MBP is considered a more reliable solubility enhancer for prokaryotic expression than GST, since dimerization of GST can promote unwanted oligomerization. Both MBP and GST are larger than most affinity tags, at approximately 42 kDa and 26 kDa, respectively. Therefore, their addition is more likely to interfere with the function of the recombinant protein. The ideal approach for selecting a fusion protein is to design multiple constructs and determine empirically which configuration promotes soluble expression in prokaryotic cells, while minimizing alterations to structure or function.
The Proteos team can take your project from inception to protein. We provide customized, adaptable, milestone-driven scopes of work to facilitate data-driven decision making while mitigating risks. Contact us today to start the discussion.
