Utilizing a protein splicing side reaction to generate a fragment for expressed protein ligation

Abstract

The intein that interrupts the DNA polymerase of Pyrococcus abyssi can facilitate protein splicing in a temperature dependent fashion. We have taken advantage of this to create a fusion protein to facilitate one-step protein purification. In short, we have fused the intein with an N-terminal affinity domain and a C-terminal target protein for purification. We bind the fusion protein to a resin, in this case a Strep-tag resin, and induce C-terminal cleavage of the intein via cyclization of Asn. Once optimized, this will release the C-terminal segment, which is a fragment of a selenocysteine-containing protein with an N-terminal Cys. This fragment will be used as a substrate in an expressed protein ligation reaction to create a semi-synthetic enzyme. This material is based upon work supported by the National Science Foundation under grant MCB-0950245 and by the Camille and Henry Dreyfus Foundation.