Abstract: The purpose of this study is to obtain a Cysteine-free intein as a biological tool for labeling or modification. Four Cysteines in HaV01 Pol were mutated and replaced by site-directed mutation and over-lap PCR, and their splicing activity was detected by Western Blot. The results showed that the 112th Cysteine negatively regulates the splicing activity of HaV01 Pol but not other three Cysteines. In addition, replacement of the 112th Cysteine with Glycine and Threonine can rescue the splicing efficiency up to 50%. Surprisingly, this is the first time to report one Cysteine localized at the unconserved block which can determine the intein′s splicing activity. In the end, two Cysteine free HaV01 Pol mutants were constructed which are facilitated for HaV01 Pol′s further transformation as well as application.

Key words: cysteine, protein splicing, site-directed mutation