Protein trans-splicing of multiple atypical split inteins engineered from natural inteins

Abstract

Protein trans-splicing by split inteins has many uses in protein production and research. Splicing proteins with synthetic peptides, which employs atypical split inteins, is particularly useful for site-specific protein modifications and labeling, because the synthetic peptide can be made to contain a variety of unnatural amino acids and chemical modifications. For this purpose, atypical split inteins need to be engineered to have a small N-intein or C-intein fragment that can be more easily included in a synthetic peptide that also contains a small extein to be trans-spliced onto target proteins. Here we have successfully engineered multiple atypical split inteins capable of protein trans-splicing, by modifying and testing more than a dozen natural inteins. These included both S1 split inteins having a very small (11-12 aa) N-intein fragment and S11 split inteins having a very small (6 aa) C-intein fragment. Four of the new S1 and S11 split inteins showed high efficiencies (85-100%) of protein trans-splicing both in E. coli cells and in vitro. Under in vitro conditions, they exhibited reaction rate constants ranging from ~1.7 × 10(-4) s(-1) to ~3.8 × 10(-4) s(-1), which are comparable to or higher than those of previously reported atypical split inteins. These findings should facilitate a more general use of trans-splicing between proteins and synthetic peptides, by expanding the availability of different atypical split inteins. They also have implications on understanding the structure-function relationship of atypical split inteins, particularly in terms of intein fragment complementation.

Figure 1. Amino acid sequences of mini-inteins and split inteins.

Mini-intein sequences are aligned using ClustalW online , and gaps (represented by -) were introduced to optimize the alignment. CneA PRP8 intein was a natural mini-intein. Ter DnaE-3 mini-intein was derived from a natural conventional split intein by a fusion of the intein fragments. Other mini-inteins were derived from natural inteins by a deletion of their putative endonuclease domain sequences, with the position and number of deleted residues shown in parenthesis. A linker sequence (ASGHHHHHHGGSGS) was inserted at the site of deletion (or corresponding site in the CneA PRP8 and the Ter DnaE-3 mini-inteins) and marked with an arrowhead. For each intein, three (or two) amino acid residues (enclosed with a rectangle) of the native extein sequences on each side of the intein were included in all splicing studies. In the Ssp DnaB mini-intein, whose crystal structure is known, sequences of the 12 β-strands (β1 to β12) are underlined. Split sites for producing the S1 and S11 split inteins are marked with black triangles.

Figure 2. Mini-intein cis-splicing in E. coli cells.

A. Schematic illustration of the cis-splicing reaction. The recombinant precursor protein consists of a maltose binding protein sequence (M) followed by the mini-intein sequence (I) and a thioredoxin sequence (T). B. Detection of splicing activity. After expression of the precursor protein containing a mini-intein (specified on top) in E. coli for overnight at 25°C, total cellular proteins were resolved by SDS-PAGE, followed by Western blotting using an anti-thioredoxin (anti-T) antibody that detected the precursor protein (MIT) and the spliced protein (MT).

Figure 3. S1 split intein trans-splicing in E. coli cells.

A. Schematic illustration of the trans-splicing reaction. Recombinant N-protein consists of a maltose binding protein sequence (M) followed by the small N-intein (I_N). Recombinant C-protein consists of the larger C-intein (I_C) followed by a thioredoxin (T). B. Detection of trans-splicing. For each S1 split intein (specified on top), the N-protein and the C-protein were co-expressed in E. coli for overnight at 25°C, total cellular proteins were resolved by SDS-PAGE, and protein bands were visualized by Coomassie Blue staining or by Western blotting using an anti-T antibody as indicated. Positions are marked for the spliced protein (MT), the N-protein (MI_N), the C-protein (I_CT), and a C-cleavage product (T).

Figure 4. S11 split intein trans-splicing in E. coli cells.

A. Schematic illustration of the trans-splicing reaction. Recombinant N-protein consists of a maltose binding protein sequence (M) followed by the large N-intein (I_N). Recombinant C-protein consists of the small C-intein (I_C) followed by a thioredoxin (T). B. Detection of trans-splicing. For each S11 split intein (specified on top), the N-protein and the C-protein were co-expressed in E. coli for overnight at 25°C, total cellular proteins were resolved by SDS-PAGE, and protein bands were visualized by Coomassie Blue staining or by Western blotting using an anti-T antibody as indicated. Positions are marked for the spliced protein (MT), the N-protein (MI_N), and the C-protein (I_CT).

Figure 5. S11 split intein trans-splicing in vitro.

For each split intein, purified C-protein (lane C) and N-protein (lane N), which are illustrated in Figure 4A, were mixed in a 1∶5 molar ratio and incubated under fixed set of conditions (25°C, 250 mM NaCl, 1 mM DTT, 20 mM Tris-HCl, pH 8.0). After specified number of minutes (specified on top), samples were taken and analyzed by SDS-PAGE and visualized by Coomassie blue staining. Positions are indicated for the precursor proteins (MIN and ICT), the splicing product (MT), and the excised N-intein (IN). The excised C-intein was too small (6 aa) to be seen in this analysis. Size markers (kD) are shown on the left.

Figure 6. Kinetic analysis of trans-splicing in vitro.

For each S1 or S11 split intein (specified in A to D), purified C-protein and N-protein (illustrated in Figures 3A and 4A) were mixed in a 1∶10 molar ratio and incubated under same conditions as in Figure 5. Samples were taken at different times (specified on top) and analyzed by Western blotting using an anti-T antibody. From the Western blot, relative amounts (band density) of the spliced protein (MT) and the C-protein (I_CT) were estimated, and the splicing efficiency was calculated as MT/(MT+I_CT). The splicing efficiency was plotted against the reaction time, which was used to estimate the reaction rate constant. All experiments were performed in triplicate, and error bars represent standard deviation.