(518g) Engineering and Characterization of a C-Terminal Cleaving Intein for Use in Protein Purification

A highly efficient C-terminal cleaving split DnaE intein from Nostoc punctiform was engineered through rational design. Npu is a thio-induced intein. A single mutation in the C-fragment of Npu, D118G, was introduced based on a sequence alignment  with the mini-Mtu recA intein from Mycobacterium tuberculosis to curb the trans-splicing reaction, giving rise to the mutant split intein Npu*. At neutral pH and in the presence of reducing agent (e.g. DTT), Npu* exhibits no detectable trans-splicing between the N- and C-exteins and instead undergoes a single cleavage reaction at the C-extein. The C-extein cleavage reaction is rapid in the presence of the N-intein and reducing agent, with ~80% cleavage occurring in just 1 hr at 37°C. To achieve the same 80% cleavage, 2 and 4 hours were needed for samples incubated at room temperature (22°C) and 16°C, respectively. We demonstrate the application of Npu* for the rapid purification of 6 proteins.