Journal: Nature

Article Title: Hijacking a bacterial ABC transporter for genetic code expansion

doi: 10.1038/s41586-025-09576-w

Figure Lengend Snippet: a , SDS–PAGE analysis of sfGFP-N150TAG expression in the presence of SisoK or G-SisoK in AI or 2-YT medium. In wild-type K12, full-length sfGFP expression yields are significantly reduced when using tryptone-containing 2-YT medium owing to competition between tryptic peptides and G-SisoK for OppA binding. sfGFP expression in 2-YT medium is recovered when using the engineered IsoK12 strain. Consistent results were obtained over three independent replicate experiments. Arrow indicates sfGFP, asterisk indicates truncated sfGFP. b , Scheme for OppA evolution to improve G-SisoK uptake in tryptone-containing medium. OppA libraries were screened for increased G-SisoK uptake under increasing tryptone concentrations by monitoring amber suppression of sfGFP-N150TAG and sorting fluorescent cells. Initial screening of an error-prone library yielded four variants, which were used as basis for creating a cassette mutagenesis library. Screening of this library identified OppA-iso. c , Extracted ion chromatograms to determine intracellular SisoK concentrations. IsoK12 cells grown in 2-YT medium show 7- to 10-fold higher intracellular SisoK concentrations compared with K12 cells when adding 2 mM G-SisoK to 2-YT medium. Consistent results were obtained over three independent replicate experiments. d , Affinity measurements of G-SisoK and a linear GSK peptide (GSK(lin)) towards wild-type OppA (wt-OppA) and OppA-iso using microscale thermophoresis. Data are mean K d values ± s.e.m. calculated from three biologically independent experiments ( n = 3). All data processing was performed using GraphPad Prism 10 (GraphPad software) and MO.affinity Analysis (v.3.0.5, NanoTemper Technologies). e , SDS–PAGE analysis of sfGFP-N150TAG expression in wild-type K12 versus IsoK12 grown in 2-YT medium in the presence of G-XisoK derivatives with X = P, Prg, ClA, C or pL. Full-length sfGFP expression is greatly increased in IsoK12 for all G-XisoK derivatives. Consistent results were obtained over three independent experiments. Asterisk indicates truncated sfGFP. Full gels and gels for other G-XisoK derivatives are presented in Supplementary Fig. .

Article Snippet: An error-prone library of opp A was generated using an error-prone PCR kit (Jena Bioscience PP-102).

Techniques: SDS Page, Analysis, Expressing, Binding Assay, Screening, Mutagenesis, Library Screening, Microscale Thermophoresis, Software

Journal: Nature

Article Title: Hijacking a bacterial ABC transporter for genetic code expansion

doi: 10.1038/s41586-025-09576-w

Figure Lengend Snippet: a . X-ray crystal structure of OppA:G-SisoK (PDB ID: 9RD1) with mutated positions highlighted as spheres. Left: Positions mutated in error prone OppA variants 1–4. Orange spheres indicate residues that were targeted for site-saturation mutagenesis; grey spheres denote additional mutations in the error prone OppA variants. Middle: Green spheres indicate residues mutated in the final evolved variant OppA-iso. Right: Table summarizing all mutations identified in error prone OppA variants 1–4 and in OppA-iso. Residues in bold were targeted for site saturation mutagenesis. b . Structural comparison of the OppA:G-SisoK complex (PDB ID: 9RD1) with the previously published structure showing OppA bound to endogenous linear peptide ligands (PDB ID: 3TCF) with a focus on binding motifs at the C-terminal end of the bound ligands. In OppA:G-SisoK, the carboxylate of lysine is in hydrogen-bonding distance to the side chains of R439, N392 and H397. In the linear peptide bound complex (OppA:XXX), the C-terminal carboxylate of the tripeptide XXX interacts primarily with the side chain of R439 and residues N392 and H397 are not ideally positioned to contribute to strong hydrogen-bonding, suggesting that the interaction with R439 is most important for linear tripeptides. In constrast, for isopeptide-linked tripeptides such as G-SisoK the hydrogen-bond donors, H397 and N392, are well positioned to interact with the lysine carboxylate, likely stabilizing the complex also in the absence of a positively charged side chain at position 439.

Article Snippet: An error-prone library of opp A was generated using an error-prone PCR kit (Jena Bioscience PP-102).

Techniques: Mutagenesis, Variant Assay, Comparison, Binding Assay