Background

Inteins (intervening proteins) are polypeptide segments translated within host proteins that are removed through a protein splicing reaction [1,2]. In the intein-mediated splicing reaction, the intein is released by the formation of a peptide bond between the flanking sequences, termed N- and C-exteins, which form the mature host protein. This reaction can proceed without any external factors, though the rate at which splicing occurs is highly dependent on the conditions present. The unique ability of inteins to rearrange peptide bonds has led to the invention of numerous applications in protein engineering [2,3]. Abundant and widespread within the microbial world [4], prior work has demonstrated that some inteins have adapted to serve as environmental sensors, displaying their capacity to regulate protein function in response to conditions such as metals, heat, salt, oxidative stress, and DNA damage [2,5,6].

To monitor intein activity in response to different conditions, protein splicing reporters are often used. These reporters are flexible in design, as their only requirement is the presence of a cysteine, serine, or threonine as the first residue following the intein [1]. Previously, we developed a kanamycin intein splicing reporter (KISR), with it providing an easy method to detect selective small molecule inhibitors of protein splicing within the cellular environment [7–10]. As inteins are absent in humans but present within human pathogens including Mycobacterium tuberculosis and Cryptococcus neoformans, splicing inhibitors may serve as novel antimicrobials, making them increasingly important targets to study [11,12]. Kanamycin A is a commonly used aminoglycoside antibiotic that inhibits translation. Bacteria with a mature aminoglycoside phosphotransferase (KanR), however, can survive in the presence of the antibiotic. The KISR reporter makes use of KanR as the exteins flanking an intein, making it so intein splicing activity confers antibiotic resistance to the experimental cells transformed with the reporter [7–10]. In the presence of small molecule inhibitors, the KISR will be unable to splice, leaving the cells vulnerable to the antibiotic. Therefore, this reporter allows for easy observation of what molecules can act as inhibitors and to what degree they inhibit, as stronger inhibitors will lead to reduced growth.

We recently developed an intein-based biosensor that adapts the KISR framework into a tripartite folding biosensor, where a protein of interest (POI) is inserted within the intein between conserved splicing blocks (Figure 1). Tripartite protein folding biosensors serve to measure the stability of a POI by linking it to reporter activity within the cellular environment [13]. Our intein-based tripartite biosensor design, while serving a similar purpose, distinguishes itself from previous biosensors through the insertion of the POI into an intein, which is then flanked with reporter extein sequences [14]. Through splicing activity, the POI is removed as the extein sequences are joined into a functional reporter molecule. This allows the biosensor to avoid the challenges associated with previous designs that kept a POI fused to the reporter, such as limitations on the size of POI insertion or restrictions on reporter function. Initial work with our biosensor was carried out using the E. coli maltose binding protein (MBP) as a POI, with it being inserted into the homing endonuclease (HEN) loop of the Pyrococcus horikoshii RadA intein (RadA-I) [14]. The HEN loop of RadA-I has been noted to accommodate POI insertion, as previous work has shown that the intein remains splicing active upon insertion of ZsGreen into the loop [15,16]. The RadA-I intein was also selected due to its rapid and accurate splicing across a variety of extein contexts [17–19], which indicated that it could accommodate reporter exteins. The RadA-I-MBP fusion was inserted within KanR (KanR-RadA-I-MBP), with kanamycin resistance serving as the reporter in a manner similar to KISR. This biosensor was then tested in a variety of conditions, where it was observed that a decrease in MBP stability (due to mutations) or inhibition of splicing activity resulted in reduced survival of cells, as they were not provided kanamycin resistance [14]. This supported the viability of our intein-based biosensor approach, as the stability of the POI was directly linked to reporter activity. Further work, which made use of the fluorescent protein TagRFP675 as the POI, showed the potential of our biosensor design as a dual-folding sensor, with both antibiotic resistance and fluorescence linked to POI stability [14]. Potential applications of our intein-based biosensor are broad due to the relaxed requirements of intein splicing, making this approach viable for linking protein stability to any desired in vivo readout [14].

Figure 1. Intein-based protein stability sensor. KanR is colored in gray, intein is colored in red, and the protein of interest (POI) is colored in orange.