Protein Retrosplicing Enabled by a Double Ligation Reaction

Proteins containing a C-terminal thioester are important intermediates in semi-synthesis. Currently there is one main method for the synthesis of protein thioesters that relies upon the use of engineered inteins. The invention involves, in some aspects a method, utilizing Sortase A, for preparation of recombinant proteins containing a C-terminal αthioester. This new method for double ligatation is useful for synthesizing new or naturally occurring molecules such as a protein thioester.