A general approach to protein folding using thermostable exoshells

Materials

Inclusion bodies of rFasxiator, α– and λ-conotoxins were obtained from Prof. Kini Manjunatha’s lab (Dept. of biological sciences, NUS). The vectors used for cloning were the pBAD/HisB vector (Life Technologies) and pRSF1b expression vector (Merck). For transformation, chemically competent BL21 (DE3) E. coli cells (catalog #RH217-J40; Simply Science) and XL1 Blue E. coli cells (catalog #RH119-80; Simply Science) were used.

Reagents

The reagents used were as follows: Kanamycin (ThermoFisher), Luria-Bertani (LB) agar (Axil Scientific), Omega bio-tek plasmid mini kit and gel extraction kit (Simply Science), Ampicillin (Axil Scientific), IPTG (Axil Scientific), L-arabinose (Sigma), Sodium Chloride (NaCl, Sigma), Tris (Sigma), Triton X-100 (Sigma), Hemin (Sigma), Calcium Chloride (CaCl₂, Sigma), Magnesium Chloride (MgCl₂, Sigma), L-glutathione oxidized (Sigma), β-mercaptoethanol (Sigma), Imidazole (Sigma), Phosphate-buffered saline (PBS, Sigma), Dithionite (Sigma), Sodium Thiosulfate (Sigma), Tween-20 (ThermoFisher), Ferrous Ammonium Sulfate (Sigma), Glycerol (Sigma), Potassium Iodide (Sigma), Coelentrazine substrate (Promega), chemiluminescent HRP substrate (Millipore), 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (ThermoFisher), Sulfuric acid (H₂SO₄, Sigma), Ethylenediaminetetraacetic acid (EDTA, Sigma), Urea (1st Base), GuHCl (Sigma), Dithiothreitol (DTT, Sigma), and Thermo ScientificTM PierceTM c-Myc Tag IP/Co-IP Kit (cat. No. 23620), sPLA₂ assay kit (Cayman), 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) (Sigma), specific Factor XIa (Merck), chromogenix S-2366 substrate (Diapharma), adenosine 5′-triphosphate (ATP) disodium salt (Sigma), D-luciferin potassium salt (Sigma), bovine serum albumin (BSA)(Santa Cruz Biotechnology), and 10X phosphate buffer saline (PBS)(Lonza). Antibodies used were as follows: mouse monoclonal anti-p53 antibody clone PAb421 (p53 Lab), mouse monoclonal anti-p53 (wild type) antibody clone PAb1620 (Sigma), mouse monoclonal anti-p53 antibody Pab240 (Abcam), and mouse monoclonal anti-hemagglutinin antibody (Sigma).

Antibodies used were as follows: concentration: mouse monoclonal anti-p53 antibody clone PAb421 (4 µM), mouse monoclonal anti-p53 (wild type) antibody clone PAb1620 (5 µM), and mouse monoclonal anti-p53 antibody Pab240 (5 µM), mouse monoclonal anti-hemagglutinin antibody (1 µg/ml).

Inclusion body POI/tES–F116H nanoparticle preparation

pBAD/HisB and pRSF1b constructs containing genes for h6POI (GFPuv, rLuc, HRPc, HSA, FFL, and PLA₂) (Genescript) and tES–F116H were transformed into chemically competent BL21(DE3) E. coli cells and grown on LB agar (Axil Scientific) plates with 50 µg/mL ampicillin and 25 µg/mL kanamycin, respectively, as reported in the previous study. For expression, a single positive colony was inoculated in 100 mL of LB broth with 100 µg/mL ampicillin and 50 µg/mL kanamycin selection. After overnight incubation at 37 °C, 12.5 mL of the starter culture was used to inoculate 500 mL of LB broth and was allowed to grow till absorbance (OD600) of 0.4 was reached. Protein expression was induced with 0.4 mM IPTG (pRSF vector) (Axil Scientific) and 0.1% L-arabinose (pBAD vector) (Sigma Aldrich) and incubated for 4 h at 37 °C. The cells were then centrifuged at 13,750 × g for 15 min.

For tES–F116H, the cell pellet was resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, and 1 mM EDTA, pH 7.5), sonicated, and then centrifuged to separate the cell debris. The supernatant was subjected to two-step chromatography purification, which included hydrophobic interaction chromatography (HIC) using HiPrepTM Phenyl FF (low sub) 16/10 (GE Healthcare) followed by size-exclusion chromatography (SEC) using a Superdex S-200 10/300 GL column (GE Healthcare). The purity of SEC fraction was analyzed using SDS-PAGE. The buffers used were as follows: HIC buffer A: 25 mM Tris-HCl, 150 mM NaCl, and 1 M (NH₄)₂SO₄, pH 7.5; HIC buffer B: 25 mM Tris-HCl, 150 mM NaCl, pH 7.5; SEC: 25 mM Tris-HCl, pH 8.0.

For h6POI, the cell pellet was resuspended in lysis buffer (25 mM Tris-HCl, 150 mM NaCl, 5 mM BME, and 1.5% Triton X-100, pH 7.5), sonicated, and centrifuged to separate the cell pellet. The cell pellet was then resuspended in washing buffers (25 mM Tris-HCl, 0.5% Triton X-100, 200 mM NaCl, 5 mM BME, pH 8.0, and 25 mM Tris-HCl, 0.5 M NaCl, 2 M urea, and 5 mM BME, pH 8.0) and centrifuged. The cell pellet was then resuspended in solubilization buffer, pH 8.0 (Table 1) to solubilize the inclusion bodies for 3–4 h (for FFL, solubilization time is 30 m), followed by centrifugation as reported previously. The supernatant was then subjected to Ni²⁺-NTA(Expedeon) chromatography purification, followed by the analysis of the eluted fraction using SDS-PAGE. Buffers used for Ni²⁺-NTA were as follows: Buffer A: Solubilization buffer, pH 8; Buffer B: Solubilization buffer, 500 mM imidazole, pH 8.

pBAD/HisB constructs containing multimeric protein genes for h6POI (AP, Omp2a) (Genescript) and Pet22b plasmid (p53 lab) were transformed into chemically competent BL21(DE3) E. coli cells and grown on LB agar plates (Axil Scientific). For expression, a single positive colony was inoculated in 100 mL of LB broth with 100 µg/mL ampicillin and incubated overnight at 37 °C for h6POI and 30 °C for p53. Following the incubation, 12.5 mL of starter culture was used to inoculate 500 mL of LB broth and was allowed to grow till absorbance (OD600) of 0.4 was reached. Protein expression was induced by 0.1% L-arabinose (pBAD) and incubated for 4 h at 37 °C. For p53, expression was induced by 0.5 mM IPTG and 0.1 mM ZnCl₂ and incubated at 30 °C for 5 h. The cells were then centrifuged at 13,750 × g for 15 min. For h6POI, the cell pellet was resuspended in lysis buffer (1.5% Triton X-100, 25 mM Tris-HCl, and 150 mM NaCl, pH 8.0), sonicated, and centrifuged to separate cell pellet. The cell pellet was then resuspended in washing buffers (0.5% Triton X-100, 25 mM Tris-HCl, 200 mM NaCl, pH 8.0, and 25 mM Tris-HCl, 0.5 M NaCl, 2 M urea, and 2 mM DTT, pH 8.0) and centrifuged. For p53, lysis and washing buffers were supplemented with 10 mM DTT. For Omp2a and AP, 10 mM β-ME was added to the lysis and washing buffers. The cell pellet was then resuspended in solubilization buffer, pH 8.0 (Table 1) to solubilize the inclusion bodies for 3–4 h, followed by centrifugation as reported previously. The supernatant was then subjected to Ni²⁺-NTA(Expedeon) chromatography purification, followed by the analysis of the eluted fraction using SDS-PAGE. Buffers used for Ni²⁺-NTA were as follows: Buffer A: Solubilization buffer, pH 8; Buffer B: Solubilization buffer, 500 mM imidazole, pH 8.

In vitro folding

The purified tES–F116H variants were acidified for shell disassembly with 25 mM Tris-citrate buffer, pH 5.8, for 30 min followed by the isolation of the subunits using size-exclusion chromatography (SEC). The monomeric POIs purified from inclusion bodies were incubated with subunits (pH 5.8) at different ratios, until the optimum tES–F116H_subunits:POI ratio was reached for maximum encapsulation efficiency. The pH of the mixture was then adjusted to 8.0 and the sample was incubated at room temperature for 30 min, followed by overnight dialysis of the mixture in folding buffer (Table 1) using Slide-A-Lyzer^® Dialysis Cassette G2 (Thermo Scientific). The folded protein encapsulated in tES was purified, the fractions acidified and subjected to a second SEC to separate the refolded proteins from the subunits. In the case of partial release of the POI from the tES, high-salt concentrations of 500 mM NaCl were used with release buffers (25 mM Tris, pH 5.8).

The refolded protein was purified using SEC. The SEC fractions were collected and checked for activity. The purification step with SEC also served as a buffer exchange to lower NaCl concentrations of 75–100 mM. The folded-protein fractions were then collected and analyzed for their functional activity. For the conotoxins, the functional fractions were analyzed using a well-characterized UPLC–QTOF mass spectrometry assay with positive standards. For the conotoxins, the pH of the mixture was adjusted to eight and incubated at room temperature for 30 min. The mixture was diluted slowly using stepwise addition of the refolding buffer, finally obtaining 10-fold dilution of the mixture. The diluted mixture was incubated overnight and then purified using SEC. The SEC fractions were collected and acidified for shell disassembly, following subjection to another SEC using 25 mM Tris-citrate, 0.5 M NaCl, pH 5.8, and buffer for separating refolded conotoxins from tES subunits. SEC elutes containing the conotoxins were analyzed using UPLC–QTOF system.

Optimal ratios of tES–F116H_subunits:POI for functional yield were determined for nine monomeric POIs. POIs were titrated against a fixed concentration of tES and after removal of denaturant and protein aggregates, when possible, the soluble fraction was quantified using individual activity assays generally performed while the monomer was still inside the shell (conotoxin and rFasxiator assays required release from the shell).

Solubilized h6POI (Omp2a, AP) were incubated in the presence of acidified disassembled tES subunits (in 25 mM Tris-HCl, pH 5.8) in optimum tES–POI molar ratio of 60:5 for maximum encapsulation efficiency. For p53, the optimum tES–POI molar ratio was 60:10 (due to near neutral net surface charge of p53). The pH of the mixture was adjusted to 8 and the sample was incubated at room temperature for 30 min, followed by overnight dialysis of the mixture in refolding buffer (Table 1) using Slide-A-Lyzer^® Dialysis Cassette G2 (Thermo Scientific). For Omp2a, dialysis was done at 37 °C, while for p53 and AP, dialysis was done at 4 °C. Following dialysis, the pH of the mixture was changed to 5.75 to disassemble the exoshell and release of refolded monomer. For p53 and AP, after release, the protein was purified using Ni²⁺– NTA chromatography and then the protein was incubated at 4 °C for 60 min to facilitate multimerization. For Omp2a, following the Ni²⁺– NTA purification, the protein was incubated at 37 °C for nine days to facilitate the multimerization.

Characterization of nanoparticles

The particle size and distribution of tES(+)F116H and tES–F116H/POI were determined by dynamic light scattering (DLS) (Nanobrook Omni, Brookhaven), using disposable cuvettes at 24 °C and the average hydrodynamic diameter was determined by taking an arithmetic average of 10 runs. The morphologies of the nanoparticles were analyzed using transmission electron microscopy (TEM, JEOL JEM-1220 TEM) and their zeta potentials were measured using Zeta-Plus analyzer (Nanobrook Omni, Brookhaven).

In vitro assays

GFPuv and HSA activities were determined through fluorescence assays, HRPc and rFasxiator activities were determined through colorimetric assays, and rLuc and FFL activities were determined through luminescence assays, respectively, from purified proteins. All reactions were performed at least in triplicates. Bar graph represents quantification as mean ± SE. Fluorescence of GFPuv and HSA was read at 508 and 340 nm in a 96-well black polystyrene plate (Fisher Scientific) with excitation wavelength fixed at 395- and 282-nm wavelength, respectively. For HSA, tES exoshells were subjected to cage break and the protein was separated from the subunits by Ni²⁺– NTA chromatography. Following the purification, the fluorescence of refolded HSA was measured. The activity of HRPc was assayed using TMB substrate in a 96-well crystal-clear polystyrene plate (Greiner Bio-One). Purified fractions were incubated in an assay buffer containing 25 mM Tris-HCl, pH 8.0, 5 mM CaCl₂, and 2.5 µM hemin for 5 min. TMB substrate was added for color development and the reaction stopped using 2 M H₂SO₄ after 5 min. Absorbance was recorded at 450 nm. rFasxiator selectively inhibits FXIa activity, thus inhibiting FXIa cleavage of Chromogenix S-2366 substrate (Diapharma). In all, 50 μl of rFasxiator was incubated with 50 μl of specific factor XIa (FXIa) (Merck) in 50 mM Tris, pH 5.8, 150 mM NaCl, and 5 mM CaCl₂ at room temperature (24 °C) for 30 min. It was followed by the addition of 50 μl of Chromogenix S-2366 substrate (3 mM) and the cleavage of the substrate was recorded at 405 nm for 4 h. All rLuc and FFL reactions took place at ambient temperature (24–27 °C) in a 96-well plate with white interior. For rLuc, luciferase activity was evaluated using Renilla luciferase kit (Promega) with some modifications. The reaction was initiated by injecting 50 µL of Renilla luciferase assay reagent (1:1,000 dilution of coelenterazine in the assay buffer). For FFL, luciferase activity was evaluated using D-luciferin potassium salt as a substrate. The reaction was initiated by injecting 50 µl of buffer containing D-luciferin potassium salt (final concentration 200 µM), 50 mM Tris-Cl, pH 7.5, 10 mM MgCl₂, 2 mM EDTA, 100 µM ATP, and 0.1% BSA, to a 50-µl protein sample. For both proteins, the assay reagents were protected from light at all times by covering the tubes with an aluminum foil. The signal was integrated for 1 min with a 2-s delay and was reported in RLU. All readings were recorded on a Perkin Elmer Plate reader. Appropriate controls were used in each case to minimize background. PLA₂ activity was measured using sPLA₂ assay kit (Cayman) in a 96-well crystal-clear polystyrene plate (Greiner Bio-One), which uses 1,2-dithio analog of diheptanoyl phosphatidylcholine as a substrate. PLA₂ hydrolyzes thio-ester bond at the sn-2 position, which releases free thiols that are detected using 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB). About 15 μl of refolded protein (in 25 mM Tris-HCl, 10 mM CaCl₂, pH 8.0 buffer) was mixed with 10 μl of DTNB, and the reaction was initiated by adding 200 μl of substrate solution to the mixture. The mixture was then incubated for 2 h, after which the absorbance was recorded at 405 nm.

Thermal stability measurement

Thermal stability of the nanoparticles was determined using a protein thermal shift assay kit (Thermofisher) in 384-well PCR plates. About 20 μl of reaction mixture containing 12.5 μl of sample, 2.5 μl of Protein thermal shift dye (8X), and 5 μl of Protein thermal shift buffer was dispensed in each well. The PCR plates were then sealed with optical seal and centrifuged to remove the air bubbles in the mixture. Thermal scanning (25–99.9 °C at 0.05 °C/s) was done using real-time PCR system (QuantStudio 7 Flex System, Applied Biosystems) and the fluorescence intensity was measured every 10 s. Analysis of the denaturation curve and determination of denaturation temperature was done using QuantStudio Real-Time PCR software (Applied Biosystems). All reactions were performed at least in triplicates. The curve represents quantification as mean ± SE.

Encapsulation-efficiency measurement

Experimentally, a Beckman Coulter ProteomeLabTM XL-I was used to determine the encapsulation efficiency of tES–F116H for different POIs. The tES–F116H/POI samples (1 µM) in PBS buffer and reference PBS buffer were loaded into the two holes of an Epon cell with sapphire windows. These cells were housed in a four-hole rotor (Ti-60, Beckman-Coulter), with one hole occupied by a reference cell for radial calibration and kept at a constant temperature of 20 °C. The optical densities of the samples at 280 nm were measured after every 7 min as a function of time and cell radius for tracking the sedimentation of the tES–F116H with and without POI encapsulation, till all the particles had sedimented to the bottom of the cells. The experiment was continued at a constant rotation speed of 40,000 × g for 7.5 h. The data were analyzed with SEDFIT and SEDNTERP software. The quality of the fit for the AUC curve was assessed using residuals plots, rmsd (<0.1), and Z-value (<15) (Tables 2, 3)¹⁴⁸.

Release of functional protein monomers and specific activity measurement

Engineered pH-responsive tES–F116H shells containing the POI—HSA, FFL, rFasxiator, PLA₂, GFPuv, HRPc, and rLuc—were subjected to cage break in 50 mM Tris-citrate, 500 mM NaCl, pH 5.8, as described above. Following the cage break, the sample was subjected to SEC and the POI activity was measured in SEC fractions. The POI was then separated from tES–F116H subunits in the eluted fractions using Ni²⁺-NTA chromatography as described above. The concentration of POI was measured in using Nanodrop (DeNovix) and POI activity was determined as described. Using POI activity and concentration values, the specific activity was calculated. tES–F116H shells containing α- and λ-conotoxins were subjected to cage break in 25 mM Tris-citrate, 0.5 M NaCl, pH 5.8, and the eluted fractions were analyzed using UPLC–QTOF systems. All reactions were performed at least in triplicates. Bar graph represents quantification as mean ± SE.

UPLC–QTOF analysis

Concentrated and pooled HPLC fractions were run on UPLC–QTOF systems (1290 Infinity II LC + Agilent 6550B QTOF). Chromatographic separation was achieved with a linear gradient using mobile phase A (0.1% formic acid in water) and B (0.2% formic acid in acetonitrile) at a flow rate of 0.40 mL/min on a Phenomenex Kinetex 2.6-μm XB-C18 100 Å 100 × 2.1 mm column with a total run time of 10 min. Electrospray-positive ionization (ESI+) mode was selected, and the mass/charge was acquired in the 100–1700 m/z range.

The chromatogram was obtained by extracting the ion count of the dominant m/z species of conotoxin (tolerating an error of 50 ppm) against acquisition time. The retention time is then compared against synthetic peptide standards to determine conformation and relative quantity of the conotoxins.

In vitro assays for multimeric POIs

Fluorescence of refolded AP was read at 340 nm in a 96-well black polystyrene plate (Fisher Scientific) with excitation wavelength fixed at 282-nm wavelength. Omp2a fluorescence was read at 351 nm with excitation wavelength fixed at 286 nm. AP activity was measured using alkaline phosphatase assay kit (Abcam) in a 96-well crystal-clear polystyrene plate (Greiner Bio-One), which uses p-nitrophenyl phosphate (pNPP) as substrate. pNPP turns yellow when dephosphorylated by AP. About 80 μl of refolded protein (1 μM in 25 mM Tris-HCl, 2 mM MgCl₂, and 4 mM ZnCl₂, pH 8.0 buffer) was mixed with 50 μl of 5 mM pNPP solution, and the plate was incubated at 25 °C for 120 min in the dark. The reaction was stopped by adding 20 μl of stop solution and the absorbance was recorded at 405 nm. For Omp2a, 0.5 ml of sample was taken after one day, three days, six days, and nine days, respectively and was subjected to size-exclusion chromatography. The eluted peak was then compared with gel-filtration markers to determine the formation of Omp2a trimer. About 12% SDS gel was used to analyze the purified multimerized p53 and Omp2a. All reactions were performed at least in triplicates. Bar graph represents quantification as mean ± SE.

Encapsulation-efficiency measurement

Experimentally, a Beckman Coulter ProteomeLabTM XL-I was used to determine the encapsulation efficiency of tES–F116H for different POIs as well as the multimerization of POIs. The tES–F116H:POI samples (1 µM) and POI (0.5 mg/mL) in refolding buffers and reference refolding buffers were loaded into the two holes of an Epon cell with sapphire windows. These cells were housed in a four-hole rotor (Ti-60, Beckman–Coulter), with one hole occupied by a reference cell for radial calibration and kept at a constant temperature of 20 °C. The optical densities of the samples at 280 nm were measured after every 7 min as a function of time and cell radius for tracking the sedimentation, till all the particles had sedimented to the bottom of the cells. The experiment was continued at a constant rotation speed of 40,000 × g for 7.5 h. AUC experiments to analyze p53-binding properties were done at a constant rotation speed of 10,000 × g for 7.5 h. The data were analyzed with SEDFIT and SEDNTERP software. The quality of the fit for the AUC curve was assessed using rmsd (<0.1) and Z-value (<15) (Tables 4–6)¹⁴⁸.

p53-binding assays

To test the folding efficiency of p53, PAb240 and PAb1620 were used. PAb1620 specifically recognizes p53 in its wild-type conformation, i.e., the conformation of the protein when it is correctly folded, by binding to the residues Arg156, Leu206, Arg209, and Gln/Asn210¹⁸⁸. PAb240 interacts with the epitope that is structurally hidden in the core of wild-type p53 (amino acids 213–217), but is exposed if the protein is denatured or misfolded¹⁸⁹. About 100 μl of antibody (5 μM) is incubated with 100 μl of purified refolded p53 (5 μM) at 4 °C on a rotor for 1.5 h to facilitate antibody binding. After incubation, the p53-antibody complex was analyzed using analytical ultracentrifuge and differential scanning fluorimetry.

Binding efficiency of p53 was analyzed using consensus p53-response element (conA) and scr-conA (jumbled conA sequence) primers (Table 7). The conA has a high binding affinity to p53, while scr-conA binds weakly or not at all to the p53 tetramer. About 100 μl of purified p53 (4 μM) was incubated with 100 μl of primers (4 μM) and 50 μl of PAb421 antibody (4 μM) at 4 °C on a rotor for 1.5 h to facilitate p53–DNA-complex formation. After incubation, the complex was analyzed using differential scanning fluorimetry and analytical ultracentrifuge.

Oligonucleotides used

Specific activities for tES-folded p53 and p53 folded alone were calculated using area under the curve for the p53 ConA complex during analytical ultracentrifugation. As no complex formation was observed using p53 folded in isolation, this ratio was qualitatively described as >100-fold.

Porin-channel measurement for Omp2a

Lipid-bilayer measurements were done by Prof. Mathias Winterhalter’s lab (Jacob’s University, Germany). Channel-recording apparatus consisted of a two-compartment Teflon chamber (~2.5 mL each) separated by a 25-μm-thick Teflon partition with an aperture of diameter ~100 μm for membrane formation (Supplementary Fig. 3). Bilayer lipid membranes were formed from 1,2-diphytanoyl-sn-glycero-phosphatidyl-choline (DPhPC) using the monolayer-opposition technique¹⁹⁰. The aperture was pretreated with a hexadecane/hexane solution (1 % v/v) and allowed to cure for ~20 min to achieve solvent evaporation. The trans– and cis-sides of the chambers were filled with buffer solution, 1 M KCl, and 10 mM HEPES at pH 7.0, 10 μL of lipid in pentane (5 mg/ml) was added to both sides of the chamber, and the bilayer is formed after evaporation of pentane. Channel reconstitution is achieved by the addition of Omp2a into the cis-side of the chamber at a volume of 0.5 μL from Omp2a stock solutions (1 mg/ml) diluted ∼10²–10⁵ times by a solution of Genapol (1.1% v/v). Channel current traces were recorded with Ag/AgCl electrodes in agarose salt bridges containing 3 M KCl, or calomel electrodes containing a salt bridge (Metrohm AG) in the case of reversal-potential measurements. The cis-side of the chamber was considered the virtual ground, and measurements were done using the Axopatch 200B (Molecular Devices, LLC) patch-clamp amplifier in V-clamp mode (whole-cell β = 1) with a CV-203BU headstage. The output signal was filtered by a low-pass Bessel filter at 10 kHz, and saved at a sampling frequency of 50 kHz using an Axon Digidata 1440 A digitizer (Molecular Devices, LLC). Data analysis is performed with Clampfit 11.0.3 (Molecular Devices, LLC), Origin Lab 2018 (Northampton, MA, USA), and self-developed LabVIEW (National Instruments) data acquisition and analysis package for acquisition and analysis of reversal-potential measurements. Both single- and multichannel-insertion analysis was done for tES–Omp2a activity. Channel insertions achieved at 20 mV and/or 50 mV. Single-channel conductance was obtained by analyzing multichannel-insertion steps ΔI/V_m = G. Multichannel insertions of tES–Omp2a were obtained at V_m = + 50 mV (positive-membrane potential) and –50mV (negative-membrane potential). Insertion frequencies of ion channels into lipid bilayers do not reflect the functional concentration of ion channels within a solution. Thus, we defined the “active” form of Omp2a as the trimeric assembly molecular weight as observed in SEC. As no trimeric formation was observed using Omp2a folded in isolation, this ratio was qualitatively described as >100-fold.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.