1. Affinity Tags for Protein Purification
Vibhor Mishra Curr Protein Pept Sci. 2020;21(8):821-830. doi: 10.2174/1389203721666200606220109.
The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed.
2. Expression and purification of soluble His(6)-tagged TEV protease
Joseph E Tropea, Scott Cherry, David S Waugh Methods Mol Biol. 2009;498:297-307. doi: 10.1007/978-1-59745-196-3_19.
This chapter describes a simple method for overproducing a soluble form of the tobacco etch virus (TEV) protease in Escherichia coli and purifying it to homogeneity so that it may be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. The protease is initially produced as a fusion to the C-terminus of E. coli maltose binding protein (MBP), which causes it to accumulate in a soluble and active form rather than in inclusion bodies. The fusion protein subsequently cleaves itself in vivo to remove the MBP moiety, yielding a soluble TEV protease catalytic domain with an N-terminal polyhistidine tag. The His-tagged TEV protease can be purified in two steps using immobilized metal affinity chromatography (IMAC) followed by gel filtration. An S219V mutation in the protease reduces its rate of autolysis by approximately 100-fold and also gives rise to an enzyme with greater catalytic efficiency than the wild-type protease.
3. Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag
Sreejith Raran-Kurussi, David S Waugh Methods Mol Biol. 2017;1607:1-15. doi: 10.1007/978-1-4939-7000-1_1.
Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His6- or a dual His6-MBP tagged fusion protein by Gateway® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His6 tag or a His6-MBP tag can be made on the basis of this solubility test.
