Evaluation and comparison of protein splicing by exogenous inteins with foreign exteins inEscherichia coli

Abstract

a b s t r a c tProtein splicing catalyzed by inteins has enabled various biotechnological applications such as protein ligation. Successful applications of inteins are often limited by splicing efficiency. Here, we report the comparison of protein splicing between 20 different inteins from various organisms in identical contexts to identify robust inteins with foreign exteins. We found that RadA intein from Pyrococcus horikoshii and an engineered DnaB intein from Nostoc punctiforme demonstrated an equally efficient splicing activity to the previously reported highly efficient DnaE intein from Nostoc punctiforme. The newly identified inteins with efficient cis-splicing activity can be good starting points for the further development of new protein engineering tools.

Intermolecular domain swapping induces intein-mediated protein alternative splicing

“…We derived the cis-splicing precursor protein (HG-I-G) from naturally split DnaE intein from Nostoc punctiforme (Npu) as a cis-splicing intein (I) flanked with HG, the N-terminally His 6 -tagged B1 domains of IgG binding protein G (GB1), as the N-extein and GB1 (G) as the C-extein 15,16 . cis-Splicing of this precursor protein produces the spliced product, HG-G 16 . The split intein precursor (I C -GH) contained the natural C-terminal split NpuDnaE intein (I C ) and the C-terminally His 6 -tagged GB1 (GH) as the C-extein.…”

“…We tested iPAS of highly efficient cis-splicing inteins, that is, RadA intein from Pyrococcus horikoshii (PhoRadA), VMA intein from Thermoplasma volcanium (TvoVMA), TFIIB intein from Methanocaldococcus jannaschii (MjaTFIIB), NpuDnaB intein and engineered MjaTFIIB and NpuDnaB mini-inteins ( Supplementary Fig. 2) 16 . We artificially created C-terminal split intein fragments from the individual cis-splicing inteins by splitting at their corres ponding sites to the naturally split site of NpuDnaE intein Supplementary Fig.…”

“…It is noteworthy that iPAS could rescue the less-active MjaTFIIB mini-intein. It has been shown that several inteins are not functional under specific conditions and are not necessarily optimized for the splicing function 16,17,31 . iPAS might have more important roles for poorly splicing and inactive inteins.…”

Recent progress in intein research: from mechanism to directed evolution and applications

“…Most inteins characterized to date are comparably slow single-turn over enzymes, and often show some level of sequence dependence at the splice junctions, which is difficult to predict and so far only very poorly understood [17,69,70]. Many biotechnological applications of inteins, however, require a traceless removal of the intein or at least a change of the flanking amino acid sequence as minimal as possible to preserve the primary sequence of the protein of interest.…”

A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein

“…Such effects of native and foreign exteins have recently been studied in detail (Cheriyan et al 2014;Oeemig et al 2012;Zettler et al 2013). A screening approach with 20 different inteins from various microorganisms has been presented, in which their functionality was investigated depending on foreign extein sequences (Ellilä et al 2011). Moreover, randomly mutated inteins were tested for splicing activity in non-native host proteins at insertion sites where the wild-type intein was shown to be inactive (Appleby-Tagoe et al 2011).…”