Streamlined expressed protein ligation using split inteins

Abstract

Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

Scheme 1. Protein Splicing (A), trans-Splicing (B) and EPL (C)

Ex^N and Ex^C represent N- and C-exteins, respectively. Int^N and Int^C represent N- and C-intein fragments, respectively.

Figure 1

Streamlined EPL using split inteins. (A) Schematic of the procedure for the isolation of an α-thioester derivative of a protein of interest (POI) using engineered split intein fragments (Int^N and Int^C). EPL can be performed in a one-pot fashion during thiolysis from the split intein or immediately after elution, without need of any further purification. (B) Sequences of Npu^C (WT) and the Npu^CAA mutant used on the split intein column. Catalytic residues mutated in Npu^CAA are shown in bold, and the linker sequence added for immobilization onto the solid support is underlined. Sequences are numbered according to the intein sequence alignment shown in Figure S2.

Figure 2

Purification of α-thioester proteins expressed in E. coli. (A) MBP, (B) PHPT1, and (C) Ub mercaptoethansulfonate (MES) α-thioesters were purified in one step from E. coli cell lysates using the Int^C column. The purifications were monitored by coomassie stained SDS-PAGE analysis (top) (inp: input, FT: column flow-through, W1-3: washes, E1-4: elutions, and bds: resin beads). RP-HPLC (detection at 214 nm) and ESI-TOF MS analysis of the eluted fractions (bottom left and right, respectively) confirmed the identity of all protein α-thioesters and indicated high purity.

Figure 3

One-pot purification/ligation of ubiquitin to the H-CGK(Fluorescein)-NH₂ peptide (CGK(Fl)). Ub-Npu^N from E. coli cell lysates was bound to the Int^C column, as shown in Figure 2, and after removal of contaminants through extensive washes, intein cleavage and ligation were triggered by addition of 200 mM MES and 1 mM CGK(Fl) peptide. Coomassie stained SDS-PAGE analysis and in gel fluorescence of the purification/ligation (left). RP-HPLC (detection at 214 and 440 nm) and ESI-TOF MS (right) of the eluted fractions confirm the desired ligated protein was obtained in one step directly from cell lysates with a ligation yield close to 95% (quantified by RP-HPLC).

Figure 4

Effect of C-terminal amino acid identity on α-thioester formation. The 20 mutants of the protein Ub-X-Npu^N were expressed in E. coli varying the identity of the C-terminal amino acid of Ub (X) from the WT Gly to all other proteinogenic amino acids. Thiol-induced cleavage yields from the Int^C column were calculated from the SDS-PAGE analysis of the eluted fractions and left over resin beads. Ratios of α-thioester vs side products were determined from RP-HPLC and ESI-TOF MS analysis of the eluted fractions. The major competing reaction for all amino acids was hydrolysis with the exception of Asn for which its succinimide form was isolated instead. * See main text for a discussion on the problems associated with Asp. Error bars ± SD (n = 3).

Figure 5

Semisynthesis of hH2B-K120Ac under denaturing conditions. (A) Coomassie stained SDS-PAGE analysis of hH2B(1–116) α-thioester generation in the presence of 2 M urea (sup: cell lysate supernatant, trit: 1% triton wash of the inclusion bodies, inp: solubilized inclusion bodies used as input for the Int^C column). E1–E3 were collected after 18 h of incubation with MES and E4–E6 after an additional 18 h. E1–E6 were pooled, concentrated to 150 μM, and ligated to the peptide H-CVTK(Ac)YTSAK-OH at 1 mM for 3 h at rt. (B) RP-HPLC (left) of the ligation reaction mixture and MS (right) of the ligated hH2B-K120Ac product.

Figure 6

Purification of a monoclonal antibody α-thioester using a split intein and its site-specific modification. (A) Test expression of αDEC205 genetically fused to the contiguous Mxe GyrA intein and different split DnaE inteins through the C-terminus of its heavy chain (HC). Western blot of 293T cell supernatants of several αDEC205-Int fusions using an antibody against mouse IgG. A loading control is shown below. (B) Purification of αDEC205-MES thioester using the split intein column. (C) Elution fractions containing αDEC205-MES were concentrated to 20 μM and ligated to the CGK(Fl) fluorescent peptide at 1 mM for 48 h at rt. (D) SEC-MALS analysis of the ligated antibody showing that it retains its tetrameric structure after thiolysis and ligation (M_w = 151 kDa, M_w calcd = 148 kDa). (E) ESI-TOF MS analysis of degycosylated and fully reduced HC after ligation, showing 75% of the HC is labeled. Expected mass for ligation product = 50221.2 Da. Free HC = 49575.0 Da.

Figure 7

Binding of αDEC205-CGK(Fl) to the DEC205 receptor. (A) Dose dependent binding of αDEC205-CGK(Fl) to CHO cells expressing the mouse DEC205 receptor monitored by flow cytometry using a PE-labeled α-mouse IgG. Binding to control CHO/NEO cells, which do not express the receptor is shown in gray. (B) As in (A) but using a control α-DEC205 antibody.