Figure 1.
Detection of trans-splicing and C-cleavage activities.
A. Schematic illustration of the C-cleavage and trans-splicing reactions. Precursor protein ICT is a fusion protein consisting of the 139-aa C-intein (IC) of the Ssp DnaX split intein fused to a thioredoxin protein (T). Precursor protein MIN is a fusion protein consisting of a maltose binding protein (M) and the 11-aa N-intein (IN) of the Ssp DnaX split intein. B. Experimental analysis of the reactions. For analysis in vivo, MIN and ICT proteins were co-expressed in E. coli cells. Total cellular proteins before (NI) and after (I) the IPTG-induced expression were resolved by SDS-PAGE, and protein bands were visualized either by staining (Coomassie stained) or by Western blotting using an anti-thioredoxin (Anti-T) antibody. For analysis in vitro, the MIN and ICT proteins were separately produced and purified. These two proteins were then mixed and incubated at room temperature for 20 hours. Reaction products were analyzed and visualized by staining or Western blotting as above. Positions are indicated for the precursor proteins (MIN and ICT), the splicing product (MT), and the C-cleavage products (IC and T). Size markers (Marker) are shown on the left. C. Effects of reaction times and temperatures. The in vitro reactions were carried out for the specified length of times and at the specified temperatures. Reaction products were analyzed by Western blotting as above.
Figure 2.
Effects of IN size and Block B mutation on trans-splicing.
A. Schematic illustration of experimental designs. INL is the N-terminal 144-aa sequence of the Ssp DnaX intein. INLm and ICm are the same as INL and IC, respectively, except that the conserved Block B sequence of the intein was mutated from TXXH to AXXA. Others are the same as in Figure 1A. B. Trans-splicing of ICT with MINL. A mixture of the two precursor proteins was incubated at the specified temperatures for 20 hours to allow reaction, with the protein bands visualized by Western blotting using anti-T antibody. C. Trans-splicing of different precursor protein pairs listed in panel A. Each pair of precursor proteins was incubated together at room temperature for 20 hours, with the protein bands visualized by Western blotting using anti-T antibody. Names of only intein parts of the precursor proteins are specified on the top of the panel.
Figure 3.
Cross-reactivity of IC with IN from different inteins.
A. Comparison of the amino acid sequences of IN from Ssp DnaX intein (IN), Rma DnaB intein (INRB), and Ssp GyrB intein (INSG). Identical and similar residues are marked with a | and a :, respectively. A gap (represented with a -) is introduced in the IN sequence to maximize the sequence alignment. B. Analysis of trans-splicing reactions between IC and INRB (or INSG) as specified on top. For in vivo analysis, MINRB (or MINSG) and ICT proteins were co-expressed in E. coli cells, and total cellular proteins were analyzed by Western blotting using an anti-thioredoxin (Anti-T) antibody. For in vitro analysis, purified MINRB (or MINSG) and ICT proteins were co-incubated at room temperature for 20 hours, and the reaction products were analyzed by Western blotting as above. Positions are marked for the precursor ICT, splicing product MT, and C-cleavage product T. Size markers are shown on the left.
Figure 4.
Structural modeling of the intein and its parts.
Computer-based modeling of the Ssp DnaX intein (A) and its parts (B, C, D) used the VMD program (http://www.ks.uiuc.edu/Research/vmd/) and the crystal structure of the Ssp DnaB mini-intein [7]. The 11-aa IN part of the intein is shown in red. The C-terminal 6-aa part of the intein is shown in blue.