Extended Data Fig. 1: Systematic screening to identify SpyCatcher003 split site and optimization of SMART-SpyCatcher.

a, SpyCatcher003 was split at various sites and the cognate pairs used to generate FLAG-SpyN^1-x-NpuN^cage-FKBP and FRB-NpuC^cage-SpyC^y-113-Myc, with x and y denoting the last and first residue of the two fragments (i.e., the split site). The isopeptide bond (formed between D117 of SpyTag003 and K31 of SpyCatcher003) is shown in sticks in the structure of SpyTag-SpyCatcher (PDB: 4MLI). The primary structure of SpyCatcher003 is shown with split sites indicated. b, Schematic of the cell-free in vitro screen used to identify the optimal split site. Conditional protein splicing (CPS) was induced either by proteolytic decaging using TEV protease or through chemically induced FKBP/rapamycin/FRB hybridization, which simulates ideal colocalization on a target cell surface. c, The result of screening FLAG-SpyN^1-73-NpuN^cage-FKBP and FRB-NpuC^cage-SpyC^74-113-Myc generated from splitting SpyCatcher003 at position 73-74. Reactions were performed with FLAG-SpyN^1-73-NpuN^cage-FKBP (1 μM), FRB-NpuC^cage-SpyC^74-113-Myc, (1 μM) and His₆-SpyTag003 (2 μM). CPS was induced by the addition of either TEV protease (10 units) or rapamycin (10 μM). The reactions were analyzed by Western blot after 24 hr incubation at 37 °C. FLAG-SpyCatcher003-Myc was used as a size standard for the spliced product; FLAG-SpyCatcher003-Myc (1 μM) reacted with His₆-SpyTag003 (2 μM) was used as a size standard for the covalent complex between the two. The legend and experimental conditions apply for subsequent panels with alterations when noted. d, Npu^cage was swapped with NrdJ-1^cage in SMART-SpyCatcher thereby giving FLAG-SpyN^1-73-NrdJ-1N^cage-FKBP and FRB-NrdJ-1C^cage-SpyC^74-113-Myc. A SpyTag003 Alexa Fluor 594 conjugate (SpyTag003-AF594) was used as the activity probe. The reactions were analyzed by Western blot. e, SpyN^1-73-NrdJ-1N^cage-FKBP with the wildtype (WT) or a catalytically dead C1A mutant of NrdJ-1N^cage was mixed with FRB-NrdJ-1C^cage-SpyC^74-113-Myc and SpyTag003-AF594. The reactions were analyzed by Western blot. f, The crystal structure of fusion NrdJ-1 (PDB: 8UBS), with sequences corresponding to NrdJ-1N and NrdJ-1C colored in cyan and orange respectively. A schematic representation of the fusion protein and the caged split intein is shown as well to indicate relevant domains in addition to their N- and C-termini and the position of the non-catalytic Cys76. g-h, A structural analysis was performed to predict the impact of introducing a C76V mutation in fusion NrdJ-1. g, The backbone of wildtype NrdJ-1 (crystal structure) and that of the NrdJ-1^C76V mutant (in silico AlphaFold2 model) are superimposable with a root mean square deviation (RMSD) of 0.56 Å. h, Residue sidechains of Cys76 and Leu2 are shown as sticks and cavities in solid grey. The experimentally determined local environment of Cys76 is shown on the left, whereas the in silico predicted local environment of C76V is shown on the right and changes between the two are indicated with arrows. i, Combinations of FLAG-SpyN^1-73-NrdJ-1N^cage-FKBP or FLAG-SpyN^1-73-NrdJ-1NC76V^cage-FKBP with FRB-NrdJ-1C^cage-SpyC^74-113-Myc or FRB-NrdJ-1C^cage(C76V)-SpyC^74-113-Myc were tested as indicated using the reaction conditions described above. The reactions were analyzed by Western blot. j, Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) characterization of the SpyCatcher003 spliced product produced by rapamycin triggered CPS between FLAG-SpyN^1-73-NrdJ-1NC76V^cage-FKBP and FRB-NrdJ-1C^cage(C76V)-SpyC^74-113-Myc. Reaction conditions followed those described above. Data shown in panels c-e, and i are representative of two independent experiments.