The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction

Abstract 

We have studied the naturally split subunit of the DNA polymerase III (DnaE) intein from Nostoc punctiforme PCC73102 (Npu) using purified proteins and determined an apparent first-order rate constant of s⁻¹ at 37°C. This represents the highest rate reported for the protein trans-splicing reaction so far ( of 60s). Furthermore, the reaction was very robust and high-yielding with respect to different extein sequences, temperatures from 6 to 37°C, and the presence of up to 6M urea. Given these outstanding properties, the Npu DnaE intein appears to be the intein of choice for many applications in protein and cellular chemistry.

Abbreviations: DnaE, subunit of the DNA polymerase III, eGFP, enhanced green fluorescent protein, gpD, bacteriophage head protein D, H₆, His₆-tag sequence, Int^C, C-terminal split intein fragment, Int^N, N-terminal split intein fragment, Npu, Nostoc punctiforme PCC73102, Ssp, Synechocystis sp. strain PCC6803, ST, Streptag II, Trx, thioredoxin

Keywords: Intein, Protein splicing, Protein ligation