Intein-mediated backbone cyclization of entolimod confers enhanced radioprotective activity in mouse models
- Published
- Accepted
- Subject Areas
- Biochemistry, Bioengineering, Biotechnology
- Keywords
- Npu DnaE intein, entolimod, cyclization, radioprotective activity
- Copyright
- © 2018 Ye et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2018. Intein-mediated backbone cyclization of entolimod confers enhanced radioprotective activity in mouse models. PeerJ Preprints 6:e26803v1 https://doi.org/10.7287/peerj.preprints.26803v1
Abstract
Background. Entolimod is a Salmonella enterica flagellin derivate. Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation. However, it caused a “flu-like” syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Cyclization is commonly used to improve the in vivo stability and activity of peptides and proteins. Methods. We designed and constructed cyclic entolimod using split Npu DnaE intein with almost 100% cyclization efficiency. We adopted different strategies to purify the linear and circular entolimod due to their different topologies. Results. After Ni-chelating affinity purification, the linear and circular entolimod were purified by size-exclusion and ion-exchange chromatography, respectively. Compared with linear entolimod, the circular entolimod showed significantly increased both the in vitro NF-κB signaling and in vivo radioprotective activity in mice. Discussions/Conclusions. Our data indicates that circular entolimod might be a good candidate for further clinical investigation.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
The base peak of digest
(A) C-entolimod is digested by trypsin (T). (B) C-entolimod is digested by chymotrypsin (CT).
The sequence coverage by T digest is
1 CFNHMWSHPQ FEKAQVINTN SLSLLTQNNL NKSQSSLSSA IERLSSGLRI 51 NSAKDDAAGQ AIANRFTSNI KGLTQASRNA NDGISIAQTT EGALNEINNN 101 LQRVRELSVQ ATNGTNSDSD LKSIQDEIQQ RLEEIDRVSN QTQFNGVKVL 151 SQDNQMKIQV GANDGETITI DLQKIDVKSL GLDGFNVNSP GISGGGGGIL 201 DSMGTLINED AAAAKKSTAN PLASIDSALS KVDAVRSSLG AIQNRFDSAI 251 TNLGNTVTNL NSARSRIEDA DYATEVSNMS KAQILQQAGT SVLAQANQVP 301 QNVLSLLRHH HHHHGS
Coverage: 98% Score: 106137
The sequence coverage by CT digest is
1 CFNHMWSHPQ FEKAQVINTN SLSLLTQNNL NKSQSSLSSA IERLSSGLRI 51 NSAKDDAAGQ AIANRFTSNI KGLTQASRNA NDGISIAQTT EGALNEINNN 101 LQRVRELSVQ ATNGTNSDSD LKSIQDEIQQ RLEEIDRVSN QTQFNGVKVL 151 SQDNQMKIQV GANDGETITI DLQKIDVKSL GLDGFNVNSP GISGGGGGIL 201 DSMGTLINED AAAAKKSTAN PLASIDSALS KVDAVRSSLG AIQNRFDSAI 251 TNLGNTVTNL NSARSRIEDA DYATEVSNMS KAQILQQAGT SVLAQANQVP 301 QNVLSLLRHH HHHHGS
Coverage: 91% Score: 8248
Combine T and CT, the coverage is 100%.