Fig. 6. In vitro modular assembly of large and highly repetitive proteins based on SasG blocks.

a Schematics of the protein assembly units showing SasG5³E² fused to the split inteins or to purification tags (Strep-tag in green, hexahistidine (H₆) tag in black). The approximate length of SasG5³E² is shown for reference⁴⁷. b SDS-PAGE analysis of E. coli BL21 clear lysates expressing the assembly units. The bands corresponding to each assembly unit are marked with “*” and the proteins’ theoretical molecular weights can be found in Supplementary Table 9. c Assembly approaches to produce SasG5¹⁸E¹². In the “one pot” assembly (left), all the cell lysates containing the assembly units are mixed and reacted together overnight (O.N.) to allow for trans-splicing with consequent release of the split inteins. The assembled protein is then obtained after two purification steps using Ni-NTA resin followed by Strep-tactin resin. In the solid-phase recursive assembly, the lysates containing the H₆-tagged unit and following assembly unit are incubated overnight with the resin to allow for protein binding to the Ni-NTA matrix and trans-splicing of the first two units. Subsequent washing steps are performed and the following assembly unit is mixed with the resin and incubated for 1 h to allow for trans-splicing. Cycles of washings and 1 h incubations with the following assembly unit are repeated until the desired product size is obtained and the final protein is eluted after the final washing step. d SDS-PAGE analysis of the resin (R) and eluate (E) from the Ni-NTA purification of the “one pot” assembly and eluate (E) after the Strep-tactin purification (Strep). The full-length final protein is indicated (SasG5¹⁸E¹²). e SDS-PAGE analysis of the Ni-NTA resin after each cycle of recursive solid-phase assembly (1–5) and final protein elution (E). The full-length final protein and other side products resulting from incomplete splicing reactions are indicated. M molecular weight marker. Protein gel images are representative of at least two independent experiments with similar results. Source data are provided as a Source Data file.