Strains and Materials

Primers used in this study were synthesized by Biolegio B.V. (Nijmegen, the Netherlands), with their sequences listed in Table S1. The Gibson Assembly Master Mix enzyme was sourced from New England Biolabs (USA), while Phusion DNA Polymerase and deoxynucleotides (dNTPs) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). DNA amplification products were purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel), and plasmid DNA from newly constructed vectors was isolated with the NucleoSpin Plasmid EasyPure kit (Macherey-Nagel). All plasmid sequences were verified by Macrogen Europe (Amsterdam, the Netherlands). Unless otherwise specified, Merck supplied the chemicals. BOOM B.V. provided Bacto Tryptone, Bacto Yeast Extract, and glycerol. Antibiotics were used at final concentrations of 50 µg/mL spectinomycin (Merck) and 100 µg/mL ampicillin (Formedium). IPTG was purchased from Thermo Fisher. For cloning, E. coli Top10 was employed, while E. coli BL21(DE3) was used for expression studies. Cultures were grown in LB broth (Formedium, Norfolk, UK) at 37 °C with 220 rpm shaking or on LB agar plates (Formedium, Norfolk, UK) unless specified otherwise.

Molecular Cloning

The tirzepatide-encoding genes were inserted into the pCDFDuet vector, which was modified to incorporate either a hybrid leader or the native OspA leader sequence. Specific primers were employed to introduce the corresponding core fragments into the vector. Due to the length of these sequences, they were divided into N-terminal and C-terminal segments before assembly. The OspR gene was incorporated into the pCDFDuet-leader-Tir-2R plasmids using the Gibson Assembly method. All plasmid constructs were verified through DNA sequencing (Macrogen Europe, Amsterdam, the Netherlands). The primers used in this study are listed in Supplementary Table S1.

Expression of Precursor Peptides Tir-2R with OspR

The pCDFDuet-Tir-2R-core plasmid was introduced into competent E. coli BL21(DE3) cells via transformation. Transformed cells were spread onto LB agar plates containing spectinomycin and incubated. Single colonies were picked and cultured in 5 mL LB broth supplemented with 50 µg/mL spectinomycin and incubated overnight at 37 °C with shaking at 220 rpm. The overnight culture was then diluted 1:50 into Terrific Broth (TB: 24 g/L Bacto Yeast Extract, 12 g/L Bacto Tryptone, 5 mL/L glycerol, 0.017 M KH₂PO₄, 0.072 M K₂HPO₄) supplemented with 50 µg/mL spectinomycin. The cultures were grown at 37 °C and 220 rpm until reaching an OD600 of 1.0–2.0. After cooling on ice, peptide expression was induced by adding 1 mM IPTG, followed by incubation at 18 °C for ~ 20 h at 200 rpm. For co-expression of Tir-2R with OspR, the induction process included 1 mM arabinose and 1 mM IPTG, while all other conditions remained identical to the peptide expression protocol described above.

Expression of Labeled Epimerized Precursor Peptides Tir-2R with OspD Using the Orthogonal D2O-Based Induction System (ODIS)

To investigate and localize epimerization by OspD, we employed the orthogonal D2O-based induction system (ODIS), originally developed by Morinaka et al. and later adapted by Korneli et al. (2021). To prepare 1 L of TB in D2O, 12 g tryptone and 24 g yeast extract were first dissolved in 900 mL D2O. The potassium phosphate buffer solution was separately prepared by dissolving 2.31 g KH₂PO₄ (0.17 M) and 12.54 g K₂HPO₄ (0.72 M) in 100 mL D2O. The two solutions were then combined and sterilized by filtration through a 0.22 μm membrane.

Precursor peptides Tir-2R, tagged at the N-terminus with a His-tag and encoded in pCDF-Duet-MCS-1, were co-transformed with pBAD-OspD into E. coli BL21(DE3). The transformed cells were plated on LB agar supplemented with 100 µg/mL ampicillin and 50 µg/mL spectinomycin. Single colonies were selected to initiate overnight cultures in 4 mL LB medium containing the appropriate antibiotics, grown at 37 °C in glass culture tubes. These starter cultures were then used to inoculate 30 mL of Terrific Broth (TB) at a 1:100 dilution, supplemented with ampicillin and spectinomycin. The cultures were incubated at 37 °C with shaking (220 rpm) until reaching an OD600 of 1.0–2.0. To induce expression of the unmodified precursor peptide, 0.1 mM IPTG was added, and the cultures were incubated at 16 °C with shaking (200 rpm) for approximately 20 h.

Following induction, cells were harvested by centrifugation at 7500 rpm for 10 min at 4 °C, and the resulting pellet was washed twice with 50 mL TB to remove residual IPTG. The washed cell pellet was then divided into two portions, both of which were resuspended in 20 mL TB supplemented with ampicillin, spectinomycin, and 0.2% (w/v) L-arabinose. One portion was resuspended in TB prepared with regular water, while the other was resuspended in TB prepared entirely with D2O. Both cultures were incubated at 16 °C with shaking (200 rpm) for an additional 20 h before peptide purification, as described in subsequent sections.

Peptide Purification

For peptide purification, cells from a 100 mL culture were collected by centrifugation (4 °C, 8,500 rpm, 5 min) and resuspended in 20 mL lysis buffer (20 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 7.4). Lysis was performed by sonication (10 s ON, 10 s OFF, 45–55% amplitude, 10–15 min). The resulting lysate was clarified by centrifugation (4 °C, 10,000 rpm, 30 min) and filtered through 0.45 μm filters. The clarified lysate was loaded onto a pre-equilibrated Ni-NTA agarose column and thoroughly mixed. The resin was washed with 10 column volumes (CV) of wash buffer (20 mM NaH₂PO₄, 300 mM NaCl, 40 mM imidazole, pH 7.4), followed by elution with 5 mL elution buffer (20 mM NaH₂PO₄, 300 mM NaCl, 500 mM imidazole, pH 7.4). To remove excess salt, the eluted sample was desalted using a PD-10 desalting column packed with Sephadex G-25 resin (GE Healthcare) and eluted in 7 mL of 50 mM Tris-HCl, pH 8.0. To release the core peptide, the His6-tagged leader was cleaved by incubating the sample with LahT150 protease (1:20 ratio) in the presence of 1 mM DTT for 2 h at 37 °C. The reaction mixture was then centrifuged (4 °C, 10,000 rpm, 15 min), filtered through a 0.45 μm filter, and subjected to a second Ni-NTA agarose purification step. The flow-through, containing the cleaved core peptide, was collected, followed by buffer exchange using 7 mL of His6-tag buffer (20 mM Tris, 300 mM NaCl, pH 7.5). Further purification was performed using a C18 open-column chromatography system (Waters). The column was first washed with 3 mL of 0.1% trifluoroacetic acid (TFA) in acetonitrile (ACN) and equilibrated with 5 mL of Milli-Q water containing 0.1% TFA. After sample loading, the column was washed with 10 mL of 15% ACN + 0.1% TFA, and the core peptide was eluted with 8 mL of 60% ACN + 0.1% TFA, followed by lyophilization. The lyophilized core peptide was stored for subsequent analysis.

The lyophilized peptide was reconstituted in Milli-Q (MQ) water and filtered through a 0.2 μm membrane to remove particulates. High-performance liquid chromatography (HPLC) purification was carried out using an Agilent 1260 Infinity HPLC system equipped with a Phenomenex Aeris™ C18 column (250 × 4.6 mm, 3.6 μm particle size, 100 Å pore size). The mobile phase consisted of acetonitrile (MeCN) and Milli-Q water, applied as a linear gradient of 45–65% MeCN over 20 min at a flow rate of 1 mL/min. Peptides, including tirzepatide-related mutants, were eluted around 55% MeCN.

MALDI-TOF Mass Spectrometry Analysis

For MALDI-TOF analysis, 1 µL of the sample was spotted onto a MALDI target plate and left to air-dry. Once dried, 1 µL of matrix solution (prepared as 5 mg/mL α-cyano-4-hydroxycinnamic acid in 50% acetonitrile with 0.1% trifluoroacetic acid) was applied on top. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was then performed using a 4800 Plus MALDI TOF/TOF Analyzer (Applied Biosystems), operating in reflector positive mode.

LC-MS/MS Spectrometry

LC-MS/MS analysis was conducted using a Shimadzu LC20 XR-series HPLC system, equipped with binary LC20ADXR pumps, interfaced with a Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific). Peptide separation was achieved using an Agilent Persuit XRs 3 C8 column (2.6 μm particle size, 100 Å pore size, Phenomenex). The column temperature was maintained at 50 °C, while the autosampler was kept at 10 °C. Samples were injected at a volume of 5 µL, with a flow rate of 0.3 mL/min.The mobile phase consisted of: Solvent A: Milli-Q water with 0.1% formic acid and Solvent B: Acetonitrile with 0.1% formic acid.

A linear gradient elution was applied as follows:0–1 min: 2% B;2–5 min: Linear increase to 45% B; 5–7 min: Linear increase to 95% B; 7–8 min: Held at 95% B; 8–8.1 min: Decrease to 0.5% B. For MS and MS/MS analyses, electrospray ionization (ESI) was performed in positive mode, with a spray voltage of 3.5 kV. Sheath and auxiliary gas flows were set to 60 and 11, respectively. The ion transfer tube temperature was maintained at 320 °C. Spectra were acquired in data-dependent mode, beginning with a survey scan from m/z 300–2000 at a resolution of 70,000, followed by MS/MS fragmentation of the top 5 precursor ions at a resolution of 17,500. Fragmentation was carried out using a normalized collision energy (NCE) of 30, with dynamic exclusion of fragmented precursor ions for 10 s. LC-MS/MS data analysis was performed using Xcalibur software.

Circular Dichroism (CD) Spectroscopy Assay

The Tir-2R-OspR and Tir-2R-OspD peptides were dissolved in Milli-Q water (MQ) to achieve an approximate final concentration of 100 µM. Circular dichroism (CD) spectra were measured at room temperature using a Jasco J-815 spectropolarimeter. The samples were placed in a quartz cuvette with a 1 mm path length, and spectral data were recorded over a wavelength range of 195–250 nm. Measurements were taken with a 1 nm bandwidth and a scanning speed of 100 nm/min, with each spectrum averaged over three accumulations. The raw CD data were baseline-corrected using Milli-Q water as a reference, and all collected data were analysed using GraphPad Prism 10.

Stability Assay in Human Plasma

Human plasma was sourced from Innovative Research Company and kept at − 80 °C until needed. Before use, the plasma was thawed at 37 °C. Tir-2R-OspR and Tir-2R-OspD were dissolved in Milli-Q water (MQ) and subsequently diluted to a final concentration of 100 µM in human plasma. The samples were incubated in a 37 °C water bath, and aliquots were collected at 1, 2, 4, 8, 16, 24, 36, 48, and 72 h. At each time point, 50 µL of the sample was withdrawn and immediately mixed with 150 µL of 100% acetonitrile to precipitate proteins. The mixtures were then centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was filtered through a 0.2 μm membrane and purified using a ZipTip with a 50 µL sample volume. The purified samples were subsequently analyzed by MALDI-TOF mass spectrometry. Each time point experiment was conducted in triplicate to ensure reliability.