Expressed Protein Ligation without Intein
Authors: Sunshine Z Leeuwon
Wesley Wei Wang
Kaci C Kratch
Yuchen Qiao
Shiqing Xu
Xiaoyan Aria Wang
Jared S Morse
Wenshe Ray Liu
Ge Yu
Journal: Journal of the American Chemical Society (2020)
Discipline: CHEMISTRY
Keywords: Proteins; C-terminus; thioester; expressed protein ligation; intein fusion
Affiliations: The Texas A&M Drug Discovery Laboratory, Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States., Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas 77843, United States., Molecular & Cellular Medicine Department, College of Medicine, Texas A&M University, College Station, Texas 77843, United States.

Abstract:  Proteins with a functionalized C-terminus such as a C-terminal thioester are key to the synthesis of larger proteins via expressed protein ligation. They are usually made by recombinant fusion to intein. Although powerful, the intein fusion approach suffers from premature hydrolysis and low compatibility with denatured conditions. To totally bypass the involvement of an enzyme for expressed protein ligation, here we showed that a cysteine in a recombinant protein was chemically activated by a small molecule cyanylating reagent at its N-side amide for undergoing nucleophilic acyl substitution with amines including a number of l- and d-amino acids and hydrazine. The afforded protein hydrazides could be used further for expressed protein ligation. We demonstrated the versatility of this activated cysteine-directed protein ligation (ACPL) approach with the successful synthesis of ubiquitin conjugates, ubiquitin-like protein conjugates, histone H2A with a C-terminal posttranslational modification, RNase H that actively hydrolyzed RNA, and exenatide that is a commercial therapeutic peptide. The technique, which is exceedingly simple but highly useful, expands to a great extent the synthetic capacity of protein chemistry and will therefore make a large avenue of new research possible. 

Retracted: No

PubPeer comments:
 This article describes a new tool (activated cysteine-directed protein ligation, ACPL) for appending various functional groups onto the C-termini of recombinant proteins without the need for intein-fusion protein systems. The main advantages of ACPL over inteins include compatibility with denaturants such as GnHCl and a larger reactivity scope with regards to residues adjacent to the Cys at site of nucleophilic substitution. I have a number of critiques about this article that I believe warrant the author s attention and can best be discussed here on PubPeer: --- 1) I would like to recognize previous work in this field that was overlooked for this publication. Specifically, I am referring to the work of Derek Macmillan and co-workers at University College London. His group have previously described the use of N- S acyl shifts with Xaa-Cys (and particularly the more reactive Gly-Cys His-Cys) sequences in recombinant proteins and peptides to generate thioesters or acyl hydrazides (refs 1, 2, and 3). Considering the conceptual overlap in generating appended functional groups to recombinant proteins, I believe citation and discussion of these articles would have been fruitful for the field and would have provided an opportunity for the authors to describe the advantages of their technique over previous work. --- 2) I have some concerns on the detail of the methods and rigor of product characterization. I applaud the inclusion of functional assays to assess their synthetic products, however much was left desired in terms of yield and purity. Unfortunately, how the mass spectrometry data was collected is not included in the main paper or supplemental. Therefore, readers do not know if the spectra presented represent the purity of the entirety of the sample, a single chromatographic peak, or a single scan from a total ion chromatogram. The yield of these products appears to be calculated based on SDS-PAGE and due to the inherently low resolution of this technique, the isolated yield cannot be established due to exclusion of side-products that do not resolve on these gels and downstream purification through Ni or desalting columns. Traditionally, one should include a higher resolution technique to asses yield and purity, such as high-performance liquid chromatography with UVvis spectrometry. If mass spectrometry were used in this case, all species present in the sample should be respresented on the mass spectrum. I would suggest the authors review the recommendations from Stephen Kent for characterization of synthetic peptides and proteins (ref 4). --- 3) Extending on the topic of rigor, I believe it is an oversight to not include a sentence on some of the side-products that can be formed in NTCB reactions, such as Lys carbamylation. This has already previously been described in the literature by David Speicher (ref 5). Of note, Speicher et al. (2004) also demonstrated cleavage with Gly in his study under similar conditions presented by the authors here and should have been cited. Furthermore, I wonder how much product is potentially lost from intramolecular cyclization due to Lys residues present on the protein of interest undergoing ACPL. --- 4) There are some small details that I would like the authors to consider as they publish in the future. One relates to the presentation of raw and deconvoluted mass spectra in this article. These are not labeled according to best practices in the field of mass spectrometry (see ref 6). The x-axes are labeled as m z (Da) - however Da is not a unit of m z (m z is unitless and represents the mass of an ion divided by the charge). For deconvoluted spectra, one should identify that the species presented are zero-charge masses calculated from a protein or peptide charge envelope. The deconvoluted spectra should be labeled with Da while raw spectra should be m z (italicized). Going back to my thoughts on assessment of products, it is also worthwhile to consider centering the deconvoluted mass on the x-axis and scaling it so that any potential side-products can be observed. I noted the authors also mentioned the following:  Since there is no commercial software for calculating protein monoisotopic peaks, we wrote a Python script to calculate all theoretical monoisotopic masses and their relative intensities for all proteins and compared them to the determined ESI-MS spectra. However there many types of computational software, both commercially and free, available for post-processing of mass spectra. This may depend on the vendor of the mass spectrometer being used however, one excellent option that accommodates many types of spectra is Unidec from the Marty lab at Univ of Arizona (see https: marty.lab.arizona.edu content software). Finally, the supplemental uses RPM as units for centrifugation - but to assist with reproducbility of experiments across labs it is best to use units of g or RCF which are invariant to the length of the rotor being used. --- References 1. Kang, J.; Richardson, J. P.; Macmillan, D., 3-Mercaptopropionic acid-mediated synthesis of peptide and protein thioesters. Chemical Communications 2009, (4), 407-409. 2. Richardson, J. P.; Chan, C.-H.; Blanc, J.; Saadi, M.; Macmillan, D., Exploring neoglycoprotein assembly through native chemical ligation using neoglycopeptide thioesters prepared via Nu2192S acyl transfer. Organic Biomolecular Chemistry 2010, 8 (6), 1351-1360. 3. Adams, A. L.; Cowper, B.; Morgan, R. E.; Premdjee, B.; Caddick, S.; Macmillan, D., Cysteine Promoted C-Terminal Hydrazinolysis of Native Peptides and Proteins. Angewandte Chemie International Edition 2013, 52 (49), 13062-13066. 4. Kent, S., Chemical protein synthesis: Inventing synthetic methods to decipher how proteins work. Bioorganic Medicinal Chemistry 2017, 25 (18), 4926-4937. 5. Tang, H.-Y.; Speicher, D. W., Identification of alternative products and optimization of 2-nitro-5-thiocyanatobenzoic acid cyanylation and cleavage at cysteine residues. Analytical Biochemistry 2004, 334 (1), 48-61. 6. Murray, K. K., Boyd, R. K., Eberlin, M. N., Langley, G., Li, L., Naito, Y. (2013). Definitions of terms relating to mass spectrometry (IUPAC Recommendations 2013), Pure and Applied Chemistry, 85(7), 1515-1609. doi: https: doi.org 10.1351 PAC-REC-06-04-06 , 
 Interesting that I received a notice from this website about comments of our paper. For comment 1, I agree it would have been nice if some previous pubs related to N to S acyl transfer were discussed. The reality is that you can not include everything. There is a page limit. Readers will have their own conclusions. Keep in mind we put together scientific publications mainly for people in the field. So sufficient background of the topic is assumed. But one critical reference was missing. It was from a Japanese colleague. It used a very similar technique to synthesize a peptide hydrazide from a peptide cysteine. I apologize for not having the name of this Japanese colleague in hand. He sent me an email to point it out. I agree that reference should have been included and will include it in our followup paper. For comment 2, we followed a routine method to characterize proteins using ESI. I believe it was 0.1% formic acid and proteins were shot into Q-exactive directly. Thermo Fisher has detailed protocols for using their instruments. We are not a specialized MS group and we did everything according to protocols. This is not a mass spectrometry paper. So fancy sample preparation and data collection and processing were not not our expertise. For comment 3, this is not a vigor issue. We did not report it simply because we did not observe it. We are a protein modification research group. We care about side reactions more than most people. For comment 4, we did not say there were no programs to process mass spectrometry data. What we said was that there was no program to calculate theoretical monoisotopic molecular weights for a protein. There is an available program isopro3 that predicts monoisotopic peaks based on simulation. A real calculation involves many looped permutations. For a protein which is large than 500 residues, it will take several days to finish the calculation using a regular desktop computer. I responded to all comments out of respect of the PubPeer s efforts for providing cross examination of published papers. But I will never do it again. Publishing a paper is already a painfully long process. Every professor has many duties such as teaching, supervising students and postdocs, serving on committees, reviewing papers and grants for others, getting their own grants, etc. This is already a hell job. Almost everyone who is productive has to commit tons of extra hours to do their jobs. Doing comment exchanges about their published papers will add another level of burden. I would like to appeal to people to make their jobs easy and let them focus on science. , 
 Hello Dr Liu - I see you have quickly addressed my comments but I think some greater time spent on your rebuttal would reduce the need for more comments on PubPeer. Indeed there are page limits that must be met, but I am not aware of stringent limits for JACS articles (vs Communications which are limited to 2200 words). In either case, references do not count towards these limits and a supplemental exists for a reason. Readers may not always have a background that perfectly encompasses the topic of the paper. My main perspective is that it is good practice to recognize work that you are building off of - particularly if the work was only just performed within the last decade. Peer review is not always perfect in this regard. For the mass spectrometry analyses, once again my only request is that more detail be provided as to what that  routine method is. What seems trivial to you is an important detail for myself and others in this field when judging how useful this technique will be, as we may want to ensure that we are not carrying forward uncharacterized side-products that can potentially impact downstream experiments. Indeed you did not observe side-reactions or report any - but my main argument is that it easy to bias yourself depending on how the data is collected and analyzed. In no way am I suggesting that fancy techniques be applied here either. Mere speculation on potential side-reactions would be of benefit. Yes, monoisotopic peaks can be calculated and simulated with existing software is what I am suggesting. If you used a ThermoFisher Q-Exactive then you should have access the available FreeStyle software which can be used to analyze raw files or simulate isotopic peaks. I ve attached an image of a simulated 1+ charge ~54 kDa Taq polymerase at 1,000,000 resolution. Obviously, it did not take many days based on how quickly I ve responded. Out of respect to readers on PubPeer and JACs, I ask that you continue to respond to comments here as science does not progress without healthy discussion. My intent is not to make your busy life any harder than it has to be, I only desire that we put forth the best scientific effort wherever possible. ![file] ,