Protein trans-splicing to produce herbicide-resistant acetolactate synthase

Abstract

Protein splicing in trans has been demonstrated both in vivo and in vitro by biochemical and immunological analyses, but in vivo production of a functional protein by trans-splicing has not been reported previously. In this study, we used the DnaE intein from Synechocystis sp. strain PCC6803, which presumably reconstitutes functional DnaE protein by trans-splicing in vivo, to produce functional herbicide-resistant acetolactate synthase II (ALSII) from two unlinked gene fragments in Escherichia coli. The gene for herbicide-resistant ALSII was fused in frame to DnaE intein segments capable of promoting protein splicing in trans and was expressed from two compatible plasmids as two unlinked fragments. Cotransformation of E. coli with the two plasmids led to production of a functional enzyme that conferred herbicide resistance to the host E. coli cells. These results demonstrate the feasibility of expressing functional genes from two unlinked DNA loci and provide a model for the design of nontransferable transgenes in plants.

FIG. 1

trans-Splicing of ALS protein in E. coli ER2744. The ALS gene carrying the herbicide resistance mutation Ala²⁶ to Val²⁶ is split by the Synechocystis sp. DnaE intein fragments (IN_n and IN_c) and is coexpressed as two inactive fusion proteins from two compatible vectors (4). Protein trans-splicing produces an active ALS protein that confers herbicide resistance to ER2744 host cells.

FIG. 2

Alignment of ALS protein sequences adjacent to the split sites. The E. coli ALSII (residues 288 to 367; accession no. S48893) and cALS (residues 357 to 445; accession no. S22490) sequences were aligned with corresponding sequences of E. coli ALSIII (residues 298 to 387; accession no. P27819) and tobacco ALSI (residues 386 to 474; accession no. P09342) and ALSII (residues 383 to 471; accession no. P09114). Residues that are identical in half or more of the proteins are highlighted. The arrowheads indicate the sites at which E. coli ALSII and cALS were split and fused to intein segments as described in the text.

FIG. 3

Immunoblot analysis of production of ALSIIm-14 by protein trans-splicing. Cells transformed with plasmids were induced with 0.3 mM IPTG for 12 h at 25°C. Whole-cell lysates from uninduced cells (lane 1) or cells expressing ALSII (lane 2), ALSIIm(N)-IN_n (lane 3), IN_c-ALSII(C) (lane 4), or ALSIIm(N)-IN_n and IN_c-ALSII(C) (lane 5) were resolved by SDS-PAGE in 12% Tris-glycine gels and immunoblot analysis using antibodies raised against the N-terminal peptide (A) or C-terminal peptide (B) of ALSII. (C) Effect of temperature on trans-splicing. Whole-cell lysates were prepared from uninduced cells (lane 1) and cells expressing ALSII (lane 2) and ALSIIm(N)-IN_n and IN_c-ALSII(C) after induction with 0.3 mM IPTG at 37°C (lane 3), 30°C (lane 4), 25°C (lane 5), and 15°C (lane 6) and were subjected to immunoblotting with antiserum against the N-terminal peptide of ALSII. The 60-kDa band in panels A and C is an unknown protein that cross-reacts with the antiserum. kD, kilodaltons.

FIG. 4

Effect of reconstitution of the ALSIIm-14 protein by trans-splicing on the growth of E. coli ER2744. (A) E. coli ER2744 transformed with plasmids expressing ALSII (sector 1), ALSIIm (sector 2), ALSIIm(N)-IN_n and IN_c-ALSII(C) (sector 3), ALSIIm(N)-IN_n (sector 4), IN_c-ALSII(C) (sector 5), or ALSIIm(N) and ALSII(C) (sector 6) were plated on M9 agar medium and incubated at 37°C (plate a), at 37°C with 100 μg of valine per ml (plate b), at 30°C with 100 μg of valine per ml (plate c), at 25°C with 100 μg of valine per ml (plate d), and at 30°C with 100 μg of valine per ml and 50 μg of SM per ml (plate e). All plates contained 0.3 mM IPTG. (B) E. coli ER2744 transformed with expression plasmids for fusion proteins as indicated on the figure were cultured in M9 medium containing 0.3 mM IPTG supplemented with valine and SM as indicated at the bottom. The cell optical density at 600 nm (OD600) was measured after incubation for 40 h at 30°C.

FIG. 5

Production of full-length cALS by protein trans-splicing. Whole-cell lysates were prepared from uninduced E. coli ER2744 cells (lane 1) or from cells transformed with plasmids expressing cALS (lane 2), cALS(N)-IN_n (lane 3), IN_c-cALS(C) (lane 4), or cALS(N)-IN_n and IN_c-cALS(C) (lane 5) and were induced with 0.3 mM IPTG for 12 h at 25°C. Immunoblots were prepared after SDS-PAGE and were probed with antibodies against the N-terminal peptide (A) or C-terminal peptide (B) of cALS. A nonspecific 60-kDa protein species was detected by the antibody against the N terminus (A). The product of trans-splicing (cALS-14) has a slightly higher molecular weight than cALS because 14 additional amino acids are inserted at the splicing junction. kD, kilodaltons.