Synthesis of non-linear protein dimers through a genetically encoded Thiol-ene reaction

Abstract

Site-specific incorporation of bioorthogonal unnatural amino acids into proteins provides a useful tool for the installation of specific functionalities that will allow for the labeling of proteins with virtually any probe. We demonstrate the genetic encoding of a set of alkene lysines using the orthogonal PylRS/PylTCUA pair in Escherichia coli. The installed double bond functionality was then applied in a photoinitiated thiol-ene reaction of the protein with a fluorescent thiol-bearing probe, as well as a cysteine residue of a second protein, showing the applicability of this approach in the formation of heterogeneous non-linear fused proteins.

Figure 1. Genetic incorporation of alkene-lysine analogs into myoglobin by the wild-type MbPylRS/PylT_CUA pair.

(A) Structures of alkenyl lysine derivatives bearing an ε-carbamate linkage (1–6), an inverted carbamate 7, an amide 8, and an urea 9. (B) Myoglobin comparative incorporation efficiencies (%) and ESI-MS results.

Figure 2. Genetic incorporation of alkene-lysine analogs 1, 2 and 3 into sfGFP.

(A) SDS-PAGE analysis of purified sfGFP. –AA: no UAA was supplemented; WT: wild-type sfGFP; 1, 2 and 3: expression in the presence of the corresponding UAA (1 mM). (B) Protein yields (*wild-type sfGFP yield is 70 mg/L, 100%) and ESI-MS results.

Figure 3. Alkenyl-sfGFP is fluorescently labeled with dansyl-thiol, and bioconjugated to lysozyme to assemble a non-linear protein dimer via the thiol-ene reaction.

(A) sfGFP bearing an alkene functionality reacts photochemically with dansyl-thiol (10) or lysozyme (LYZ). (B) SDS-PAGE analysis demonstrates the labeling of alkenyl-sfGFP with 10 after 5 min of UV irradiation via thiol-ene ligation (lanes 5 and 6). Fluorescence (top) and Coomassie stain (bottom). (C) SDS-PAGE analysis shows mobility band shifts from 28 kD to 44 kD after samples were UV irradiated for 10 min (lanes 8 and 9), corresponding to the molecular weight of sfGFP-lysozyme conjugate. WT: wild-type sfGFP; 1 and 2: sfGFP carrying the corresponding UAA; LYZ: lysozyme. –UV: samples were not exposed to UV irradiation. +UV: samples were irradiated at 365 nm for 5 or 10 min.