Abstract
Protein splicing is an autocatalytic molecular process that ligates the N- and C-terminal flanking sequence, called “extein”, and liberates “intein” domain itself. This process comes in two flavors. Splicing reaction in single polypeptide from one component in terms of cis-splicing compared trans-splicing that associated two components by using split inteins. The biological application of protein trans-splicing (PTS) such as site-specific incorporation of biophysical probes, segmental isotope labeling for NMR structure or dynamics research, protein semi-synthesis containing posttranslational modifications, and protein cyclization. Protein trans-splicing also elicits a possibility from segmental isotope labeling and macromolecular protein structure determination by solution NMR, base on the Nostoc punctiforme (Npu) dnaE split intein scaffold, we successfully express the target membrane protein by two separated complementary pieces and ligate the fragments in vitro based on the trans-splicing function of split intein. Besides, we also noticed that Npu split intein has great tolerance in the presence of either detergents or denaturants. In this study, we divided full-length outer-membrane protein X (OmpX) into two fragments and successfully ligated in 4 Molar urea conditions. The ligated product refolded in DHPC detergent and checked by gel shift assay. The related NMR experiment is currently ongoing. We believe this strategy can be applied to large membrane proteins in near future.