Abstract: Inteins are protein splicing elements that excise themselves from precursor protein. It has been widely used to purify recombinant proteins. In the current study, using the Impact-Twin System to express the fusion protein CBD-Ssp DnaB intein-C-extein, researchers investigated the first C-extein residue that may influence the cleavage of Ssp DnaB intein. The results showed that cleavage of the fusion precursor protein was temperature and time dependent and was optimal when pH was 7.0-7.5. Efficiency of cleavage was also, dependent on the first residue of C-extein as well as time and temperature. The data may help in elaborating a facile method for producing other proteins of interest.