Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources

Abstract

Inteins excise themselves out of precursor proteins by the protein splicing reaction and have emerged as valuable protein engineering tools in numerous and diverse biotechnological applications. Split inteins have recently attracted particular interest because of the opportunities associated with generating a protein from two separate polypeptides and with trans-cleavage applications made possible by split intein mutants. However, natural split inteins are rare and differ greatly in their usefulness with regard to the achievable rates and yields. Here we report the first functional characterization of new split inteins previously identified by bioinformatics from metagenomic sources. The N- and C-terminal fragments of the four inteins gp41-1, gp41-8, NrdJ-1, and IMPDH-1 were prepared as fusion constructs with model proteins. Upon incubation of complementary pairs, we observed trans-splicing reactions with unprecedented rates and yields for all four inteins. Furthermore, no side reactions were detectable, and the precursor constructs were consumed virtually quantitatively. The rate for the gp41-1 intein, the most active intein on all accounts, was k = 1.8 ± 0.5 × 10(-1) s(-1), which is ∼10-fold faster than the rate reported for the Npu DnaE intein and gives rise to completed reactions within 20-30 s. No cross-reactivity in exogenous combinations was observed. Using C1A mutants, all inteins were efficient in the C-terminal cleavage reaction, albeit at lower rates. C-terminal cleavage could be performed under a wide range of reaction conditions and also in the absence of native extein residues flanking the intein. Thus, these inteins hold great potential for splicing and cleavage applications.

FIGURE 1.

Schemes of the protein trans-splicing pathway and of the constructs used in this study. A, following intein fragment association, an N–S or N–O acyl shift forms a thioester or oxoester bond at the N-extein/intein junction. This reactive intermediate is attacked in a trans(thio)esterification by the side chain sulfhydryl or hydroxyl group of the first residue in the C-extein, which can be Cys, Ser, or Thr, to give a branched intermediate. The cyclization of the conserved Asn residue at the C terminus of the intein releases the intein. Finally, the (thio)ester bond between the exteins rearranges to a peptide bond by a spontaneous S–N or O–N acyl shift. In general, X can be sulfur or oxygen. Note that all four new inteins characterized here employ a Cys-1 and a Ser+1 as catalytic residues at the splice junctions. B, shown are the intein fusion proteins used in this study and the products of the protein trans-splicing (left panel) and the trans-cleavage reactions (right panel). ST-gpD-5aa^N is the Extein^N and 5aa^C-Trx-H₆ is the Extein^C. 5aa^(N/C) represent the 5 native extein amino acids of the respective intein flanking the intein domain N- or C-terminally.

FIGURE 2.

Analyses of protein trans-splicing reactions. Shown are Coomassie Brilliant Blue-stained SDS-gels (4–12% acrylamide). In all cases, the precursor proteins (indicated by numbers) were converted into the SP and the excised intein fragments Int^N and Int^C. Samples at t = 0 min are precursor proteins that were boiled before mixing. A, time-dependent analysis of the gp41-1 intein reaction performed at 45 °C. M denotes molecular marker. B, analyses of the reactions involving the gp41-8, NrdJ-1m and IMPDH-1 inteins performed at 37 °C. The band marked with an asterisk is a protein contamination. C, time-dependent analysis of the Npu DnaE intein reaction performed at 37 °C. The band marked with an asterisk denotes a protein impurity, whereas the band marked with a number sign corresponds to the N-terminal cleavage product (ST-gpD) resulting from cleavage of the linear thioester by β-mercaptoethanol in the SDS-sample buffer used to quench the reaction (as observed previously in Ref. 26).

FIGURE 3.

Time courses and temperature dependence of protein trans-splicing reactions. A, shown are constant rates as a function of temperature. Data for the Npu DnaE intein are included for comparison. B, time courses of SP formation catalyzed by the gp41-1 intein at different temperatures. C, time courses of splice product formation for the gp41-1 as well as gp41-8, NrdJ-1, and IMPDH-1 inteins at their optimal temperature as indicated and pH 7. Error bars in panels A–C indicate S.D.

FIGURE 4.

C-cleavage with and without native extein amino acids. For these reactions at 37 °C, a C1A mutation was introduced into each Int^N fusion protein. 5 native C-extein amino acids (5aa^C) were either included (left panels) or deleted (right panels). A–D, shown are Coomassie Brilliant Blue-stained SDS-gels. The C-terminal precursor proteins (indicated by numbers) were either completely or partially converted into the Extein^C (indicated as Trx in all cases) and the Intein^C fragment. The N-terminal precursor protein remained unchanged during the reaction. Proteins bands marked with an asterisk denote protein impurities. M denotes molecular marker. E, C-cleavage constant rates of the four inteins determined at 25 and 37 °C. Error bars indicate S.D.