Cloning, protein production and purification
RAD and MBH genes were obtained from IDT and GenScript, respectively (sequences can be found in Supplementary Table 1). All genes were cloned into a vector containing a hexa-histidine-tag and TEV-cleavage site using Golden Gate assembly with a BsaI restriction enzyme54. The plasmid sequence is available at https://doi.org/10.5281/zenodo.15494922. Enzymes for amplification, mutagenesis and cloning were sourced from New England Biolabs.
For initial screening, the plasmids were transformed into Escherichia coli BL21 (DE3) STAR cells using standard procedure (30 min incubation on ice, 30 s heat shock at 42 °C, and regeneration in SOC medium at 37 °C, 1,000 rpm). A 96-well plate containing 500 µl of LB medium with 50 µg ml−1 kanamycin in each well was inoculated with a single colony for each design, including a positive control, a negative control and a sterile control (sequences in Supplementary Table 1). Cultures were grown overnight at 37 °C, 250 rpm. One hundred microlitres of these cultures was used to inoculate 24-well plates containing 3 ml of ZY autoinduction medium. For each sample, two replicates were performed. Cultures were grown at 37 °C, 200 rpm for 6 h, then at 18 °C, 200 rpm for 18 h.
Samples were collected by centrifugation (20 min, 4,800g) and resuspended in lysis buffer (20 mM sodium phosphate (sodium phosphate), 500 mM NaCl, 1% n-octyl-β-D-glucopyranoside, 20 µg ml−1 DNase I, 250 µg ml−1 lysozyme, 1× cOmplete protease inhibitor pill per 100 ml, pH 7.4). Lysis was performed at room temperature for 1 h on a vibrating plate at 1,000 rpm. The lysate was clarified (20 min at 4,800g), and the supernatant was purified in a 96-well plate using magnetic Ni-NTA beads and an Opentrons OT-2 pipetting robot. The lysate was incubated with the magnetic beads for 20 min at room temperature, and the supernatant was discarded. Beads were washed with 200 µl wash buffer (20 mM sodium phosphate, 500 mM NaCl, 2 mM tris(2-carboxyethyl)phosphine (TCEP), pH 7.4) once. The supernatant was discarded, and bead-bound proteins were eluted by adding 100 µl elution buffer (20 mM sodium phosphate, 500 mM NaCl, 250 mM imidazole, 2 mM TCEP, pH 7.4) twice per well, yielding 200 µl eluate for each replicate. Protein concentration was determined via Bradford assay (bovine serum albumin standard curve). Protein purity was confirmed using SDS–PAGE.
Active site lysine-to-alanine variants were generated by site-directed mutagenesis using primers designed with the NEBaseChanger tool. PCR reactions contained 10 µl of 5× NEB Q5 buffer, 32.5 µl ddH 2 O, 1 µl of dNTP mix (10 mM in ddH 2 O), 2.5 µl of forward and reverse primers (10 µM in ddH 2 O), 1 µl of template DNA (2 ng µl−1) and 0.5 µl Q5 DNA polymerase. Reactions were cycled for 25 rounds (98 °C denaturation, primer-specific annealing temperature, 72 °C extension), with initial denaturation (30 s) and final extension (2 min) steps. A complete list of primers and annealing temperatures used can be found in Supplementary Table 9. To 1 µl of the PCR reaction, 1 µl each of T4 DNA ligase buffer, T4 DNA ligase, T4 polynucleotide kinase, and DpnI as well as 5 µl of ddH 2 O were added and incubated at 22 °C for 1 h. Plasmids were transformed into E. coli TOP10 cells and plated on agar plates (50 µg kanamycin per ml agar). Plasmids from single colonies were isolated using a Monarch Spin Plasmid Miniprep kit and sequence-verified. Screening of lysine-to-alanine variants was performed in the same way as the original designs.
For batch production, 10 ml of TB medium containing 100 mg l−1 kanamycin was inoculated with a single colony of BL21 (DE3) STAR cells containing the respective plasmid. After overnight growth at 37 °C, 140 rpm, 10 ml of the culture was used to inoculate 1 l TB medium (same antibiotic). Cultures were grown to an OD 600 of 0.6–0.8 at 37 °C and 140 rpm, and induction was initiated by adding isopropyl β-d-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. Cells were collected 4–5 h after induction via centrifugation (20 min, 4,000g). For MBH19-63, cultures were incubated at 20 °C, 140 rpm overnight following induction and collection the next morning. Pellets were washed once with 30 ml of 0.9% NaCl solution at room temperature and stored at −20 °C.
Pellets were thawed and resuspended in lysis buffer (20 mM sodium phosphate, 500 mM NaCl, and a spatula of DNase I and lysozyme per 200 ml, pH 7.4). The suspensions were sonicated for 15 min on ice and the lysate was centrifuged at 43,000g for 40 min. The supernatant was loaded onto gravity columns containing 1–2 ml nickel immobilized metal affinity chromatography (Ni-IMAC) resin equilibrated with lysis buffer and washed with wash buffer (see above). The purified proteins were eluted using an elution buffer (see above). Buffer was exchanged to storage buffer (20 mM sodium phosphate, 300 mM NaCl, 2 mM TCEP, pH 7.4 for RAD designs; 20 mM sodium phosphate, 150 mM NaCl pH 7.4 for MBH designs) using centrifugal filters. For RAD designs, His-tag cleavage was performed by adding 0.062 mg of TEV protease (produced in-house) per mg of protein and incubation at 4 °C overnight. The cleaved tag was removed using reverse Ni-IMAC. MBH His-tags were not removed. The final purification step consisted of gel filtration on a S75 Increase 10/300 GL or S75 10/300 column equilibrated with the respective storage buffer. Protein concentrations were determined by specific absorbance at 280 nm and Bio-Rad assay. Samples were flash-frozen in liquid nitrogen and stored at −80 °C.
Intact mass spectrometry
Five microlitres of protein samples (10 μM) in RAD storage buffer were desalted on a Shim-pack Scepter C4-300 (G) column (3 μm) by washing with 1% methanol in the presence of 0.1% formic acid. Increasing concentrations of acetonitrile (MeCN, 1–95%) with 0.6% formic acid eluted the proteins into an Impact II ESI-Q-TOF (Bruker) mass spectrometer. Protein signatures were integrated and deconvoluted using the DataAnalysis maximum entropy function.
Circular dichroism
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