Peer review of: Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
In this paper, Yao and colleagues report the development of a method to detect protein-protein 
interactions (PPIs) based on the trans-splicing reaction mediated by the split intein gp41-1. The 
authors justify why they decided to develop such method saying that the current methods have 
several limitations, for instance the inability to detect weak and transient interactions or to 
function in all cellular compartments. While I do agree that good methods to robustly detect PPIs 
are very important, I fail to see how the method presented here represents the solution to the 
problem. 
 
Weak and transient interactions can be detected to date either using complementation of 
fluorescence (BiFC) or transcriptional readouts. In both cases, transient interactions are converted 
in a long-lasting response (either fluorescence which, unless specifically engineered to be labile, is 
quite stable, or a gene product). Few years ago, a method called SPARK was developed that 
combines a transcriptional readout with light activation, so that even PPI dynamics can now be 
tracked. The authors do not cite the SPARK method (Kim et al., elife, 2017), however in my eyes 
this method is more powerful than the one described here. It is also compatible with high-
throughput and can be used for drug screening. 
 
BiFC-based methods can be adapted to detect PPIs in almost all cellular compartments, provided 
the FP used is able to fluoresce also in oxidizing conditions. The SIMPL method presented here is, 
also in this respect, not the solution to the problem, since, in my opinion it would fail for proteins 
in oxidizing compartments such as ER and mitochondria because inteins contain cysteines which 
would form disulfide bonds in oxidizing compartments (see also comment below about the 
experiment in mitochondria). 
 
Beyond these points, my strongest criticism is that I think inteins are not suited for this type of 
assay. Inteins are difficult to work with because they are poorly folded (the C-intein being 
completely disordered and the N-intein being partially disordered; Shah et al., JACS, 2013). When 
fused to other proteins, they often lead to poor expression of the fusion proteins. Moreover, the 
splicing reaction is entirely dependent on the amount of N- and C-intein fragments. Changes in 
expression levels of either the bait or prey constructs would lead to very different results, 
compromising the reliability of the conclusion regarding the interaction. Here the authors had to 
split the intein at a different position to decrease the affinity between the two fragments, which 
otherwise would give rise to a huge background. This also leads to lower splicing efficiency. This 
likely compromises the ability to detect PPIs at physiological protein expression levels. 
 
Taken together these considerations, I do not feel it appropriate for this paper to be published in 
Nature Communications. 
 
Below are further comments to the authors. 
 
 
1. The authors should directly start by saying that they had to mutate the intein in order to have 
little to no affinity between the two fragments. It is highly misleading otherwise, because inteins 
are used in bioengineering to bring together proteins/peptides that have nothing to do with each 
other! I think that experiments with the wild type intein should be omitted or used as negative 
control, but cannot by any means represent the first dataset. 
 
2. Decreasing the affinity between intein fragments has been done in the past to control the trans-
splicing reaction with light (Tyszkiewicz and Muir, Nature Methods, 2008). 
 
3. Inteins are small, this is true, but they are highly problematic when it comes to folding issues. 

Even when fusing them to otherwise properly folded proteins they create problems and lead to 
very low expression levels of the fusion proteins. The authors should mention this problem as 
several PPIs may not be detected because the prey and bait (one or both) may not be expressed 
properly when in fusion with the intein fragments. This can be seen directly in the data shown in 
Fig. 1c, where it is clear that the FRB-IntN fusion is poorly expressed in comparison to the IntC-
FKBP1A fusion. 
When expressed together, the intein fragments can bind and this stabilizes the proteins. I think 
this is the reason why the authors get such a large amount of spliced product when using 
rapamycin. Indeed, since the two intein fragments work in a 1:1 ratio, the spliced product can be 
only as much as the least abundant between the IntN and IntC. The authors should discuss this in 
the paper, as it may be very confusing for readers otherwise. 
 
4. In the Western blots, loading controls should be shown. This is standard practice. Moreover, in 
this specific case, it is very important to convince the readers that the spliced product is not visible 
in the absent of interaction not because less sample was loaded on the gel, but because the 
splicing did not occur. 
 
 
5. I had a hard time deciphering the schematics. I strongly suggest the authors to simply 
represent their constructs with the various components from N- to C-terminus. There is no 
problem in writing something like: V5-IntN-Bait. 
 
6. In Fig. 2b, I cannot reconcile the bands shown for the NIN-FRB+IC-FKBP1A sample with what 
they should be. In my opinion, the IN and NIN constructs should have the same size, and this 
should approx. be 22 kDa. Thus, in the lane in which only NIN-FRB is shown, the band should run 
below 25 kDa, just like it does for the IN-FRB sample, shown in the first lane. Same problem for 
the spliced product: according to my calculations, it should be approx. 15 kDa (indeed, Bait-V5-
FLAG and Prey-V5-FLAG should be very similar in size, thus the band corresponding to the product 
should run at a very similar size as the product for the FRB-IN+FKBP1A-CIC). For this calculation, 
I considered the following sizes: FRB: 11 kDa, IN (100 aa): 11 kDa, IC (25 aa): 3 kDa, FRB-IN: 22 
kDa, FKBP1A: 12 kDA, IC-FKBP1A: 15 kDa, FRB-FKBP1A (considering also the various linkers, 
which however should not add more than a couple kDa to the entire construct): 26-27 kDa. 
 
7. I like the fact that the authors thought of problems that might arise when tagging a protein of 
interest to the “wrong” terminus. However, eventually they still did not consider all possible 
scenarios: what if the bait tolerates only N-terminal tagging and the prey only C-terminal tagging? 
The problem of the small size of the spliced product (only the V5+FLAG tags according to the 
current design) could be easily overcome by adding some other protein instead of the tags, be it 
GFP, MBP or TRX. 
I think the authors should add one such scenario to the paper for completeness. 
 
8. In Fig. 3c: the blue curve seems to suggest that there is quite some signal in the absence of 
rapamycin. Would that mean that one would actually think the bait and prey interact when in fact 
they don’t? Indeed, for me this is a critical point about the paper: split inteins really like to interact 
and, despite the fact that the new splice site reduced the affinity of the two inteins (and also their 
splicing ability, judging by the amount of N- and C-fragments still remaining on the blots in all 
cases…), I still believe the background is non-negligible and when expression levels are high 
enough, false positives may be a true problem. The authors, therefore, should show what kind of 
signal they get in the absence of prey all together. 
 
9. In the ELISA set-up: the authors normalize their signal after splicing to the signal of the bait. 
However, splicing will be affected by the amount of both, prey and bait. If in the various conditions 
(e.g. different rapamycin concentrations), different amount of prey is used, the final readout could 
be very misleading. 
As far as I understood from the method section, in Fig. 3c cells were independently transfected 

with the constructs and treated with the different doses of rapamycin. My concern is that 
transfections lead to very inhomogenous expression levels. Not knowing the amount of the prey is 
a problem somehow, because it makes it difficult to judge the final product. 
Splicing is highly dependent on the concentrations of the intein fragments… 
Indeed, what are the physiological levels of the various proteins tested in this paper? Would the 
interaction be detected were the levels very low? 
On the opposite hand of the spectrum: what if bait and prey are both expressed at too high levels? 
The authors should show titrations of their constructs and the resulting splicing. 
 
 
10. In Fig. 3f: did all bait give a signal? Or did the authors encounter expression problems in some 
cases? 
Moreover, is a PPI considered positive if positive in any of the other methods or in all of them? 
 
 
11. Fig. 4a, b: also here, loading controls must be included to convince the readers that splicing 
did not occur in the absence of interaction. 
 
12. Fig.4c,d: The authors emphasize that their method is able to detect PPIs in various cellular 
compartments. However, I do have some difficulties in believing that the splicing effectively 
occurred in the mitochondria, because of their oxidizing nature. Inteins have been shown not to 
work under oxidizing conditions due to the formation of disulfide bonds (Aranko et al., PLoS ONE, 
2009). Therefore, what I think it’s happening is that the reaction is taking place after cell lysis. 
Indeed, the data shown here are from an ELISA assay, that is, cells were lysed, thus 
compartmentalization was lost. In Material and Methods, it is mentioned that cell lysates were 
incubated for 3h at 4C. I wonder whether in this time, the intein fragments had the time to meet 
and react, thus the spliced product would not necessarily reflect an interaction that occurred inside 
the compartment. I suggest the authors to do this experiment: they should mis-localize one of the 
baits or preys, for instance by adding an NES and having the protein in the cytosol. The 
corresponding partner should instead reside in the inner mitochondrial membrane or the matrix, as 
normal. Splicing should not occur in this case, unless it happens when the proteins meet after cell 
lysis. 
 
 
 
Minor points 
 
1. In Fig. 1d and e, HEK 293 should be written on top of the image to be consistent with the other 
panels in the same figure. 
 
2. In Fig. 1f and g, the molecular weights on the side of the image are missing. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
Yao et al. describe a clever method termed SIMPL to detect protein-protein interactions by fusing 
two proteins to the N- and C-terminal fragments of an intein. Binding of the two proteins 
reconstitutes the intein, which results in a splicing event that fuses the two proteins into a single 
polypeptide. This (or another fused) polypeptide can be detected by a mobility shift on a Western 
blot. Alternatively, an ELISA assay can be used that takes advantage of two epitope tags that 
become fused together after intein activity, one for protein capture and one for detection by an 
antibody coupled to horseradish peroxidase. 
 
While the method is likely to prove useful, the manuscript has a couple of major limitations. One, 

the data presented largely consist of testing known interactions and comparing their detection to 
several other methods. Some of the comparison methods are applicable, like SIMPL, only to 
defined protein pairs that can be used to construct tagged versions, while others, like yeast two-
hybrid or protein complementation assays, can be applied to detect previously unknown 
interactions using library screens. Thus, given that the manuscript’s most common readout for 
defined protein pairs is a Western blot – a non-trivial amount of work – and in the absence of 
novel results with the SIMPL assay, it is difficult to judge whether the method will achieve broad 
acceptance and usage. 
 
The second concern is the lack of, or unclear, quantification of the results in the manuscript. 
Signals on Western blots are described with terms like “relatively high level,” “hardly detected,” 
“no obvious signal,” “effectively detected,” “successfully detected” etc. For an experimenter 
attempting this method and observing weak bands in the spliced position that is indicative of 
protein interaction, this kind of terminology is unlikely to be of much use in determining if an 
interaction has occurred. Can some kind of standard be applied and defined for users? 
 
In the case of the ELISA readout, for which quantification is provided, the manuscript can be 
cryptic. Supplemental Table 2 provides specific quantitative data, but these are aggregated in 
Figure 3f as “positive” or “negative.” More useful in Figure 3f might be a heatmap that indicates 
the relative degree of signal. It would also be helpful to have the ELISA results roughly calibrated 
to signals on the Western blots. Further, in Figure 3c, one of the arrangements of the intein tags 
(red line) maxes out in luminescence upon protein interaction (highest level of rapamycin) near 
the basal level without an interaction (no rapamycin) of one of the other arrangements (purple 
line). How does one decide which arrangement of intein domains to use without testing multiple 
combinations? 
 
The data for the high AUC values for the reference datasets of protein interactions for the SIMPL 
assay are not sufficiently described. For example, “positive” SIMPL results range from RLU means 
of 3.97 down to 0.58, with about half of these positives having a value <1.0. An additional 14 
pairs, 6 of which are labeled as false positives, are at least 80% of the low end 0.58 value. Here 
again, a user of the assay who will not want to reconstitute and retest the whole reference set of 
data would struggle to interpret intermediate RLU values. I could find no clear statement in the 
text of what constitutes “detection” of an interaction by SIMPL. 
 
In Supplemental Figure 3, many RLU values are shown as positive SIMPL signals, all the way down 
to <0.125. Is this the same scale as Supplemental Table 2? Are these values provided after 
subtraction of background? 
 
Minor concerns 
One concern is that the manuscript is more promotional than might be appropriate. I found 
statements like “SIMPL displays an excellent ability to follow the kinetics…”; “these results clearly 
demonstrate the potential for using SIMPL as a highly sensitive…tool”; “SIMPL is an exceptionally 
sensitive method”; “an unprecedentedly high sensitivity” etc. to be opinions of the authors that 
might be toned down in favor of more objective statements. 
 
What is a “regular” protein detection method (Abstract and line 124). 
 
Fig. 2a could be simplified, especially the inverted letters labeling the domains, to better convey 
the arrangements.