Background

Phylogenetically widespread in the microbial world, inteins (intervening proteins) have generated substantial interest as agents of autocatalysis, drivers of evolution, antibacterial and antifungal targets, and for biotechnological application (Lennon and Belfort, 2017). In the protein splicing reaction, the intein is removed by rearranging two peptide bonds and connecting the surrounding sequences, known as N-and C-exteins, with a peptide bond. While this process can occur in the absence of any external factors, its rate can be highly variable depending on conditions. Recently, compelling evidence has demonstrated that inteins can act as environmental sensors that regulate protein function in response to conditions such as metals, heat, salt, oxidative stress, and DNA damage (Belfort, 2017). Thus, significant interest from both basic and applied communities necessitate the investigation of inteins.

The study of intein catalysis is most often achieved using protein splicing reporters. The only strict requirement of these reporters is the presence of a cysteine, serine, or threonine as the first residue immediately following the intein (Mills et al., 2014). We have utilized a reporter, referred to as MIG (MBP-Intein-GFP), to study the activity of a variety of inteins (Topilina et al., 2015a and 2015b; Kelley et al., 2016 and 2018; Lennon et al., 2018; Green et al., 2019; Woods et al., 2020). In the MIG reporter, the intein (surrounded by 10 native extein residues) is flanked by the Maltose Binding Protein (MBP) as the N-extein and superfolder GFP (sfGFP) as the C-extein (Figure 1). Products with sfGFP attached are detected using in-gel fluorescence immediately following SDS-PAGE. sfGFP-tagged products (those containing a C-extein) allow for the monitoring of all possible intein reactions, including splicing (resulting in fluorescent ligated exteins), N-terminal cleavage (resulting in fluorescent intein-C-extein), and C-terminal cleavage (resulting in fluorescent C-extein) (Figure 1). Depending on the intein and reaction conditions, not all products may be detected. The major advantages of our sfGFP-based reporter, compared to other protein splicing reporters, is that there is no need for purification following expression and products can be visualized without staining.

Figure 1. Maltose binding protein-Intein-sfGFP (MIG) protein splicing reporter. Products of splicing, N-terminal cleavage, and C-terminal cleavage that are visible (i.e., sfGFP-containing) using in-gel fluorescence are shown for each reaction.

Moreover, the use of in-gel fluorescence is broadly applicable beyond the study of protein splicing. This in-gel fluorescence protocol can be used to accurately examine the size of any sfGFP-tagged protein expressed in E. coli without purification. For example, this strategy could be used to study the localization of proteins in different cellular fractions, such as the cytoplasm, periplasm, and membrane of E. coli. Similar procedures have been described for visualization of GFP-tagged proteins. The protocol described herein differs as it allows visualization without staining (Pristov et al., 2015) and does not require running gels at 4°C (Bird et al., 2015). Several critical parameters relating to sfGFP-detection are also addressed. As GFP structure is required to maintain its fluorescent signal, it is critical that samples are not heated prior to SDS-PAGE (Figure 2). Further, we find that the use of Tris-Glycine rather than Bis-Tris gels improves sensitivity (Figure 2).

Figure 2. sfGFP detection within SDS-PAGE. Tris-Glycine gels (Panel A) are more sensitive than Bis-Tris gels (Panel B) for detecting in-gel sfGFP signals as indicated by stronger signal, lower background, as well as detection of the minor product (IC) only in Tris-Glycine gels. sfGFP fluorescence is specifically detected in cell supernatants without purification. Heating samples at 95°C for 5 min results in a loss of fluorescent signal. Left, Coomassie stained. Right, sfGFP signal. Expected molecular weights of products are as follows: Precursor (P) 88.5 kDa; Ligated exteins (LE) 74.0 kDa; Intein-C-extein (IC) 42.6 kDa.