Self-cleaving fusion tags for recombinant protein production

Abstract

Fusion expression is a common practice for recombinant protein production. Some fusion tags confer solubility on the target protein whereas others provide affinity handles that facilitate purification. However, the tag usually needs to be removed from the final product, which involves using expensive proteases or hazardous chemicals and requires additional chromatography steps. Self-cleaving tags are a special group of fusion tags that possess inducible proteolytic activity. Combined with appropriate affinity tags, they enable fusion purification, cleavage and target separation to be achieved in a single step, which saves time, labor and cost. This paper reviews currently available self-cleaving fusion tags for recombinant protein production. For each system, an introduction of its key characteristics and a brief discussion of its advantages and disadvantages is given.

Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

“…A specific advantage of split inteins in this respect is that uncontrolled in vivo cleavage is circumvented, which may have caused poor yields of the target protein (39). We generated a C1A mutant for each of the four novel split inteins to test whether these proteins could be used for C-terminal cleavage applications.…”

Technology advancements in antibody purification

“…Site-specific cleavage is initiated by either low-molecular-weight compounds or by a conformational change in the fusion protein. 49 Examples of self-cleaving tags include intein, sortase-A, N-terminal protease, Fc-regulated protein C (FrpC), and cysteine protease domain. 30,50 Inteins are self-splicing proteins that are capable of excising themselves from a translated protein.…”

“…30 The intein cleavage tag was used many times for protein purification; however, its use has been confined to laboratory scale, mainly because the intein tag does not promote protein solubility or protein expression but merely acts as an affinity tag. 49 In addition, it is thought that the intein tag may cause uncontrolled cleavage, thus lowering protein yield. 49 In the "Thermoresponsive elastin-like polypeptide tag" section of this review, a description of the use of an intein self-cleaving tag for the isolation of target protein in a temperature-dependent purification using elastin-like protein (ELP) is given.…”

“…49 In addition, it is thought that the intein tag may cause uncontrolled cleavage, thus lowering protein yield. 49 In the "Thermoresponsive elastin-like polypeptide tag" section of this review, a description of the use of an intein self-cleaving tag for the isolation of target protein in a temperature-dependent purification using elastin-like protein (ELP) is given. 29 FrpC is an alternative self-cleavage tag from Neisseria meningitidis that is capable of mediating Ca 2+ -dependent self-cleavage.…”

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant

“…This technique allows for selection of OMV subpopulations at the purification level, since only vesicles containing the His-tag will adhere to the resin and be purified. The technique can be further advanced through incorporation of a cleavable linker that will allow purified OMVs to be restored to their native composition for use in further experimental procedures in which the presence of a His-tag may impact results [31]. For incorporating this purification technique into a pharmaceutical downstream OMV processing scheme, it may be beneficial to incorporate the His-tag via homologous recombination to ensure that all secreted OMVs possess the affinity tag.…”