Highly efficient protein trans‐splicing by a naturally split DnaE intein from Nostoc punctiforme

Abstract

Protein trans-splicing by the naturally split intein of the gene dnaE from Nostoc punctiforme (Npu DnaE) was demonstrated here with non-native exteins in Escherichia coli. Npu DnaE possesses robust trans-splicing activity with an efficiency of >98%, which is superior to that of the DnaE intein from Synechocystis sp. strain PCC6803 (Ssp DnaE). Both the N-and C-terminal parts of the split Npu DnaE intein can be substituted with the corresponding fragment of Ssp DnaE without loss of trans-splicing activity. Protein splicing with the Npu DnaE N is also more tolerant of amino acid substitutions in the C-terminal extein sequence.

Recognition of the disordered p53 transactivation domain by the transcriptional adapter zinc finger domains of CREB-binding protein

“…Segmentally labeled p53TAD-TAZ1 constructs were generated using the Nostoc punctiforme PCC73102 (Npu) DNA polymerase III (DnaE) intein system (75)(76)(77). p53TAD was expressed with an N-terminal H 6 GB1 tag and was fused to the N terminus of the N-split of the DnaE intein (Fig.…”

Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site‐specific chemical modification

“…Protein trans-splicing system has opened many applications including segmental isotopic labelling of proteins, protein cyclization, in vivo protein engineering, and site-specific chemical modifications [6][7][8][9][10][11][12][13][14][15]. However, protein ligation by protein trans-splicing can be significantly modulated by the junction sequences as well as by the extein sequences, which could limit the applications of protein trans-splicing [16]. Fusion proteins with artificially split intein fragments often become insoluble because of the unstructured split fragments [6,17].…”

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

“…Even though the previously reported DnaE intein is more tolerant of variations in the splicing junctions, it cannot splice with all possible sequences at the splicing junctions [4]. It is of practical importance to identify more inteins with robust splicing activity with various junction sequences for the development of biotechnological tools, which could alleviate the limitations imposed by the junction sequences [2,4]. Currently, more than 550 genes have been registered in the intein database (InBase) based on their characteristic sequence motif [8].…”

“…High efficiency of protein splicing is desirable to maximize yields of protein ligation by PTS, particularly for multi-fragment ligation such as central fragment isotopic labeling [6,7]. Even though the previously reported DnaE intein is more tolerant of variations in the splicing junctions, it cannot splice with all possible sequences at the splicing junctions [4]. It is of practical importance to identify more inteins with robust splicing activity with various junction sequences for the development of biotechnological tools, which could alleviate the limitations imposed by the junction sequences [2,4].…”

“…This protein trans-splicing (PTS) has great potential to manipulate primary structures of proteins in vivo as well as in vitro because protein splicing does not require any additional energy or co-factors [3]. However, low splicing efficiency and poor tolerance of sequence alternations near the splicing junctions, which are insufficiently characterized for most of inteins, hinder wider application of inteins [4,5]. High efficiency of protein splicing is desirable to maximize yields of protein ligation by PTS, particularly for multi-fragment ligation such as central fragment isotopic labeling [6,7].…”