Enzyme-Catalyzed Expressed Protein Ligation

Protein semisynthesis;PTEN;Subtiligase;Protein engineering;Phosphorylation;Signalling;Phosphatase;not listed

Henager, Samuel HCole, Philip A ;

Johns Hopkins University

关键词: Protein semisynthesis;    PTEN;    Subtiligase;    Protein engineering;    Phosphorylation;    Signalling;    Phosphatase;    not listed;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60221/HENAGER-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Expressed protein ligation involves the chemoselective reaction of recombinant protein thioesters produced via inteins with N-Cys containing synthetic peptides and has proved to be a valuable method for protein semisynthesis. Expressed protein ligation requires a cysteine residue at the ligation junction which can limit its use. Here we employ subtiligase, a re-engineered form of the protease subtilisin, to ligate a range of synthetic peptides, without the requirement of an N-terminal cysteine, to a variety of recombinant protein thioesters in rapid fashion. We have further broadened the scope of subtiligase-mediated protein ligations by employing a second-generation form (E156Q/G166K subtiligase) and a newly developed form (Y217K subtiligase) for ligation junctions with acidic residues.We have applied subtiligase-mediated expressed protein ligation to the generation of tetraphosphorylated, monophosphorylated, and non-phosphorylated forms of the tumor suppressor lipid phosphatase PTEN. In this way, we have demonstrated that the natural sequence around the ligation junction produced by subtiligase rather than cysteine-mediated ligation is necessary to confer the dramatic impact of tail phosphorylation on driving PTEN;;s closed conformation and reduced activity. We thus propose that subtiligase-mediated expressed protein ligation is an attractive traceless technology for precision analysis of protein post- translational modifications.