Manipulation of protein splicing side‐reactions to facilitate protein purification and expressed protein ligation

Abstract

The PolII intein from Pyrococcus abyssi can be modified by mutation to facilitate cleavage of either the flanking N‐extein or C‐extein in a temperature dependent fashion. Taking advantage of cleavage at the intein N‐terminus, we have created a fusion protein with a target protein at the N‐terminus, followed by the intein and a C‐terminal His tag. The fusion protein is bound to an affinity resin via its C‐terminus, and cleavage is induced such that the N‐terminal target protein is highly purified in one step. We have also designed a fusion protein that takes advantage of the C‐terminal cleavage side‐reaction. This fusion protein has an N‐terminal affinity domain, followed by the intein and the C‐terminal target protein. The fusion protein is bound to an affinity resin via its N‐terminus, and the C‐terminal segment is cleaved and purified. As this C‐terminal segment may have an N‐terminal Cys, the purified protein can serve as a nucleophile for expressed protein ligation.This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.