Protein thioester synthesis enabled by sortase.
Proteins containing a C-terminal thioester are important intermediates in semisynthesis. Currently there is one main method for the synthesis of protein thioesters that relies upon the use of engineered inteins. Here we report a simple strategy, utilizing sortase A, for routine preparation of recombinant proteins containing a C-terminal (α)thioester. We used our method to prepare two different anthrax toxin cargo proteins: one containing an (α)thioester and another containing a D-polypeptide segment situated between two protein domains. We show that both variants can translocate through protective antigen pore. This new method to synthesize a protein thioester allows for interfacing of sortase-mediated ligation and native chemical ligation.
Department of Chemistry, Massachusetts Institute of Technology, 16-573a, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Jingjing J
JJ
Ling
Rocco L
RL
Policarpo
Amy E
AE
Rabideau
Xiaoli
X
Liao
Bradley L
BL
Pentelute
D019881:Aminoacyltransferases / Q000378:metabolism*; D000942:Antigens, Bacterial / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004952:Esters / Q000737:chemistry*; D047668:Inteins; D010455:Peptides / Q000138:chemical synthesis; D021381:Protein Transport; D011506:Proteins / Q000138:chemical synthesis*; D011994:Recombinant Proteins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013457:Sulfur Compounds
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004952:Esters; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D013457:Sulfur Compounds; C030325:anthrax toxin; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases