Enzyme-catalyzed expressed protein ligation.
Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-terminal cysteine (N-Cys)-containing peptides, but the requirement of a Cys residue at the ligation junction can limit the utility of this method. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN.
Department of Pharmacology and Molecular and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Samuel H
SH
Henager
Department of Pharmacology and Molecular and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Nam
N
Chu
Department of Pharmacology and Molecular and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Zan
Z
Chen
Department of Pharmacology and Molecular and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA.
David
D
Bolduc
Department of Pharmacology and Molecular and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Daniel R
DR
Dempsey
Department of Pharmacology and Molecular and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Yousang
Y
Hwang
Department of Pharmaceutical Chemistry and Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.
James
J
Wells
Department of Pharmacology and Molecular and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Philip A
PA
Cole
D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D015153:Blotting, Western; D020134:Catalytic Domain; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D051059:PTEN Phosphohydrolase / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics
D010446:Peptide Fragments; D011994:Recombinant Proteins; D051059:PTEN Phosphohydrolase; D013381:Subtilisins; D010453:Peptide Synthases; C117282:subtiligase; D003545:Cysteine
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