Ultrafast protein splicing is common among cyanobacterial split inteins: implications for protein engineering

Abstract

We describe the first systematic study of a family of inteins, the split DnaE inteins from cyanobacteria. By measuring in vivo splicing efficiencies and in vitro kinetics, we demonstrate that several inteins can catalyze protein trans-splicing in tens of seconds rather than hours, as is commonly observed for this autoprocessing protein family. Furthermore, we show that when artificially fused, these inteins can be used for rapid generation of protein α-thioesters for expressed protein ligation. This comprehensive survey of split inteins provides indispensable information for the development and improvement of intein-based tools for chemical biology.

Figure 1

Trans-splicing of split DnaE inteins. (a) Scheme depicting protein trans-splicing of the KanR protein with a variable local C-extein sequence. (b) In vivo relative trans-splicing efficiencies at 30°C with the endogenous “CFN” C-extein sequence and exogenous “CGN”, “CEN”, and “CRN” sequences. IC₅₀ values (± SE, n = 3-4) are normalized to the intact KanR proteins with the corresponding tri-peptide.

Figure 2

In vitro trans-splicing reactions. Indicated split intein pairs fused to model exteins Ub and SUMO (Ub-Int^N and Int^C-SUMO) were mixed at 30 °C or 37 °C, and the formation of products was monitored over time by gel electrophoresis. (a) Half-lives were extracted from the reaction progress curves fit to a first-order rate equation (± SE, n = 3). Coomassie-stained SDS-PAGE gels showing (b) fast Ava splicing at 37°C and (c) inefficient Ssp splicing at 37°C.

Figure 3

Sequence-activity relationships in split DnaE inteins. (a) Inteins in order of in vivo splicing activity with selected slices from the corresponding multiple sequence alignment. (b) Rendering of the Npu structure highlighting the proximity of position 120 to the terminal catalytic residues C1 and N137. (c) In vivo analysis of the C120G mutation in the Aha intein (± SD, n = 3). (d) Rendering of the Npu structure highlighting key catalytic residues (orange sticks) and important non-catalytic positions (green spheres) that modulate Ssp activity. (e) In vivo analysis of Ssp-to-Npu point mutations that improve Ssp activity (± SD, n = 4). Note, all residue numberings correspond to the relevant positions on Npu as defined by the NMR structure (PDB: 2KEQ).

Figure 4

Engineered versions of DnaE inteins support efficient expressed protein ligation. (a) Scheme showing the formation of the linear thioester intermediate and its use to generate a protein α-thioester for EPL. (b) Coomassie-stained SDS-PAGE gel depicting MESNa thiolysis of ubiquitin from a fused AvaDnaE intein to yield the Ub-MES thioester, 4. (c) Fluorescent SDS-PAGE gels showing the formation of the Ub-CGK(Fluorescein) ligated product (6) from one-pot thiolysis and native chemical ligation reactions using the inteins indicated. (d) RP-HPLC chromatographs showing pH dependence of precursor amide (1) and linear thioester (2). A third minor peak is indicated by *.