Figure 1
From: Modular Protein Ligation: A New Paradigm as a Reagent Platform for Pre-Clinical Drug Discovery
Development of Modular Protein Ligation. (a) Conceptualization of EPL. The protein of interest (POI) is expressed as C-terminal intein fusion. The genetically modified intein (catalytic Asn mutated to Ala to block final excision step) catalyzes an N-S acyl shift, which in the presence of excess thiol, results in the release of the intein and the formation of a thioester at the C-term of the POI. The thioester undergoes attack from an N-term cysteine linked to a respective functional moiety (termed R group) and undergoes a spontaneous S-N acyl shift to form a stable peptide bond between the POI and the cysteine-containing peptide/protein fragment. (b) Conceptualization of MPL. Moving clockwise from the POI-intein fusion: (I) Cys-R, where R represents a functional moiety such as lanthanide binding peptide, 10xHis, dual STREP, etc.…, (II) free C-terminus of POI generated in presence of excess DTT, (III) POI with Cys-[K-(PEG)n], (IV) POI with Cys-[K-PEG2-Biotin], or (V) POI with Cys-[K-Fluorescein].
