Functional characterization of a naturally occurring trans-splicing intein from Synechococcus elongatus in a mammalian cell system

Abstract

We have cloned and characterized a naturally occurring split mini-DnaE intein capable of protein trans-splicing in the cyanobacterium Synechococcus elongatus (Sel DnaE intein). Sel DnaE intein is homologous to Synechocystissp. PCC6803 (Ssp) DnaE intein and Nostoc punctiforme (Npu) DnaE intein, with a protein sequence identity of 60% for the N-terminal part of intein and 61% for the C-terminal part of intein. Our results demonstrate that the split reporters, split Renilla luciferase (Rluc) and enhanced green fluorescent protein (EGFP), can be reconstituted via Sel DnaE intein-mediated trans-splicing in mammalian cells. Based on Sel DnaE intein-mediated reconstitution of split Rluc, a human immunodeficiency virus (HIV) entry-mimicking cell-cell fusion assay was developed and validated as a useful assay for screening and pharmacologically characterizing potential HIV entry-targeting inhibitors.