A Novel Tandem-Tag Purification Strategy for Challenging Disordered Proteins

Abstract

Intrinsically disordered proteins (IDPs) lack well-defined 3D structures and can only be described as ensembles of different conformations. This high degree of flexibility allows them to interact promiscuously and makes them capable of fulfilling unique and versatile regulatory roles in cellular processes. These functional benefits make IDPs widespread in nature, existing in every living organism from bacteria and fungi to plants and animals. Due to their open and exposed structural state, IDPs are much more prone to proteolytic degradation than their globular counterparts. Therefore, the purification of recombinant IDPs requires extra care and caution, such as maintaining low temperature throughout the purification, the use of protease inhibitor cocktails and fast workflow. Even so, in the case of long IDP targets, the appearance of truncated by-products often seems unavoidable. The separation of these unwanted proteins can be very challenging due to their similarity to the parent target protein. Here, we describe a tandem-tag purification method that offers a remedy to this problem. It contains only common affinity-chromatography steps (HisTrap and Heparin) to ensure low cost, easy access and scaling-up for possible industrial use. The effectiveness of the method is demonstrated with four examples, Tau-441 and two of its fragments and the transactivation domain (AF1) of androgen receptor.

Improved Expression of Aggregation-Prone Tau Proteins Using a Spidroin-Derived Solubility Tag

“…Other solubility tags have been used in the Tau field to improve the solubility of recombinant Tau proteins produced using a bacterial expression system [41,70]. However, the NT* solubility tag has the competitive advantage of enhancing the solubility of recombinant proteins with relatively low solubility and substantially boosting the overall yield of the purified protein.…”

“…Tobacco etch virus (TEV) protease, Thrombin, and SUMO protease (Ulp1) are the three most used proteases in the cleavage of solubility and affinity tags from the purified protein of interest [38][39][40]. A prime example of where a solubility tag played a pivotal role in improving the solubility of IDPs, including full-length Tau and some of its domain constructs, is our newly developed tandem-tag system (6xHis-SUMO-Tau-Heparin) [41]. Despite producing protein preparations of very high purity, the overall yield was limited for use in downstream applications like NMR titration experiments and biophysical protein-protein interaction studies.…”

Per Aspera ad Chaos: Vladimir Uversky’s Odyssey through the Strange World of Intrinsically Disordered Proteins

“…The collection of papers and reviews in this section embodies this philosophy to study IDPs. This appears as one of the most intense fields of study, with 6 scientific articles and one review [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ] ( Table 1 ).…”