Abstract

Split inteins have been used as a versatile tool in protein engineering to mediate efficient in vivo and in vitro trans-splicing of a protein. The trans-splicing ability of split inteins was also applied to the in vivo cyclization of a protein. However, cyclization efficiency is dependent upon the type of split inteins employed and the conditions under which cyclization occur. In this study, a novel reporter system that easily measures the cyclization efficiency of split inteins was developed. For this purpose TEM-1 β-lactamase was divided into two fragments (24~215 and 216~286 amino acids) and circularly permuted. The circularly permuted β-lactamase expressed in Escherichia coli showed little β-lactamase activity, most likely due to the structural modification of the protein. However, when the circularly permuted β-lactamase was cyclized by the Synechocystis sp. PCC6803 DnaB split mini-intein, β-lactamase activity both in vitro and in vivo was recovered. These results suggest that the novel reporter system can be exploited to develop new inteins with high efficiency of in vivo protein cyclization.

• Keywords: protein cyclization; split intein; circularly permuted β-lactamase