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Expressed Protein Ligation Without Intein

preprint
submitted on 09.01.2020, 16:02 and posted on 10.01.2020, 10:01 by Yuchen Qiao, Ge Yu, Xiaoyan Wang, Kaci C. Kratch, Wesley Wei Wang, Jared S. Morse, Sunshine Z. Leeuwon, Wenshe Liu
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 approach with the successful synthesis of ubiquitin conjugates, ubiquitin-like protein conjugates, histone H2A with a 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.

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History

Email Address of Submitting Author

wliu@chem.tamu.edu

Institution

Texas A&M University

Country

United States

ORCID For Submitting Author

0000-0002-7078-6534

Declaration of Conflict of Interest

No.

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in Journal of the American Chemical Society

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