Abstract
Interferon-stimulated gene 15 (ISG15) is a member of the ubiquitin-like modifiers (ULM) family, which adopts a β-grasp fold domain(s) similar to ubiquitin (Ub) with only minor sequence homology. ISG15 consists of two Ub-like domains and aids the immune system in neutralizing infections by numerous pathogens and plays an important role in defending cells against many viruses including influenza A. Recently, Ub was found to be a substrate for ISG15, which can be ISGylated on Lys29 and Lys48, while the former is more dominant. The discovery of such hybrid ISG15-Ub chains brought forward various fundamental questions regarding the nature and effect of this conjugation. To further investigate the role of hybrid ISG15-Ub chains, the pure homogeneous material of these chains is needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the total chemical synthesis of a 231-residue ISG15-Lys29-Ub hybrid chain. During the synthesis we encountered insoluble peptide fragments, and therefore we developed a new reversible Acm based solubilizing tag to efficiently tackle this hurdle. This new Acm tag was compared with the known Arg based Acm solubilizing tag and was found to be more reliable in terms of incorporation and efficiency as demonstrated in the synthesis of the native ISG15-Ub hybrid chain.
Highlights
Ubiquitin (Ub) is a 76 amino acid protein involved in many cellular events, e.g., trafficking, transcription, and proteasome degradation.[1,2] The covalent attachment of Ub to a Lys residue of a target protein requires three enzymes, E1, E2, and E3, and is known as ubiquitination.[3]
We envisioned the total chemical synthesis of the ISG15Lys29-Ub protein equipped with a biotin tag, providing a useful tool for further studies
After analyzing the sequence of Interferon-stimulated gene 15 (ISG15), made of 157 residues, we initially decided to divide it into three shorter peptide fragments, which can be prepared by Fmoc-SPPS
Summary
Ubiquitin (Ub) is a 76 amino acid protein involved in many cellular events, e.g., trafficking, transcription, and proteasome degradation.[1,2] The covalent attachment of Ub to a Lys residue of a target protein requires three enzymes, E1, E2, and E3, and is known as ubiquitination.[3] Repeating this cycle on an ubiquitinated protein produces multiubiquitination either on different Lys residues or on the same site, eventually leading to different cellular signals.[4,5] On the other hand, ubiquitin-like modifiers (Ubls) are a family of proteins similar in structure to Ub, yet different in sequence. A key to our success was the development and application of a new Acm derivative to serve for cysteine protection and increase the peptide solubility
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
