GoKawiilExpression and purification of SC003-mi3 VLP
The mi3-VLP (also known as SC003-mi3) was derived from i3-01 (ref. 66) by introducing two mutations that remove surface-exposed cysteines to prevent aggregation and by genetically fusing a SpyCatcher domain to the N-terminus to enable plug-and-play attachment of SpyTagged proteins54. The plasmid pET28a-SpyCatcher003(SC003)-mi3 (gift from Professor Mark Howarth, Cambridge University) was transformed into Escherichia coli BL21(DE3) RIPL cells (Agilent) and plated on Luria-Bertani (LB) agar supplemented with 50 µg ml−1 kanamycin. After incubation for 16 h at 37 °C, a single colony was used to inoculate 10 ml LB media containing 50 µg ml−1 kanamycin and grown overnight at 37 °C with shaking at 200 rpm. This starter culture was transferred into 1 l LB medium with the same antibiotic and incubated at 37 °C, 200 rpm, until the OD600 reached about 0.6. Protein expression was then induced with 0.42 mM IPTG and cultures were grown for a further 16 h at 22 °C with shaking (200 rpm). Cells were collected by centrifugation at 4,000 × g for 15 min. Cell pellets were resuspended in 40 ml lysis buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8.5 at 4 °C) containing 0.1 mg ml−1 lysozyme, cOmplete EDTA-free protease inhibitor cocktail (Roche, 1 mg ml−1) and 1 mM PMSF. The suspension was passed through a high-pressure homogenizer (Constant Systems) at 30,000 psi, with 2–3 passes on ice. The lysate was clarified by centrifugation at 35,000 × g for 45 min at 4 °C and the supernatant was collected. Ammonium sulfate was then added at 170 mg/ml of lysate and the mixture was incubated at 4 °C for 1 h with agitation (220 rpm) to precipitate the particles. Following centrifugation at 30,000 × g for 35 min at 4 °C, the pellet was resuspended in 8 ml buffer (25 mM Tris-HCl, 150 mM NaCl, pH 8.0 at 4 °C) and passed through 0.22-µm filters (Croning). The filtrate was dialysed overnight against a 500-fold excess of the same buffer at 4 °C. Dialysed material was centrifuged at 17,000 × g for 30 min at 4 °C to remove insoluble aggregates and filtered again (0.22 µm). Purification was completed by size-exclusion chromatography on a HiPrep Sephacryl S-500 HR 16/60 column (GE Healthcare) equilibrated in 25 mM Tris-HCl, 150 mM NaCl, pH 8.0 at 4 °C, using an ÄKTA Pure 25 system (GE Healthcare). Elution was performed at 1 ml min−1, collecting 1-ml fractions. Fractions containing SpyCatcher003-mi3 nanoparticles were pooled, concentrated and dialysed into TBS 25 mM Tris-HCl, 150 mM NaCl, pH 8.0 at 4 °C using a 100-kDa MWCO centrifugal filter (Millipore) and stored at −80 °C. Final protein concentration was determined by BCA assay (Pierce, Thermo Fisher Scientific).
Solution self-assembly experiments
It was shown previously66 that mi3-VLPs can reversibly disassemble and reassemble at guanidinium thiocyanate concentration of 2.5 M. To perform the assembly experiments, we started with a solution containing purified mi3-VLPs (Supplementary Fig. 1) at an mi3-monomer concentration of 84 μM and disassembled the VLPs using 2.5 M of guanidinium thiocyanate at varying mi3-monomer concentrations ranging from 35 to 5.8 μM. Following an incubation time of 30 to 60 min at room temperature, assembly was initiated by rapidly diluting the protein solution 100-fold in assembly buffer (20 mM HEPES pH 7.4, 138 mM NaCl) to a final mi3-monomers concentration ranging between 350 and 58 nM. The assembly reactions equilibrated at room temperature for an extra 30 min. We quantified the distribution of masses of the reassembled VLPs at the chosen concentrations using a standard MP landing assay.
MP measurements in solution
The equilibrated assembly reactions (Fig. 1 and Supplementary Figs. 2 and 3) were measured using a commercial mass photometer (TwoMP, Refeyn Ltd.) using an imaging field of view of 4.3 × 10.9 μm2. Measurements were conducted on microscope glass coverslips (24 × 50 mm, Menzel Gläser, VWR 630-2603) that were pre-cleaned by three consecutive 5-min cycles of bath sonication in acetone, 50% isopropanol in Milli-Q water (18.2 MΩ cm) and Milli-Q. Cleaned coverslips were then dried using nitrogen flow and 3-mm silicone gaskets (GBL103250, Grace Bio-Labs) were attached to the coverslip surface. The gasket was prefilled with 15 μl of buffer and the focus position was adjusted for maximum contrast before adding 5 μl of protein solution. Measurements were performed at a frame rate of 500 Hz followed by frame binning of 2, resulting in an effective frame rate of 250 Hz. We analysed data using DiscoverMP v2024R1 (Refeyn Ltd.), in which rolling ratiometric videos were generated using an averaging window size of 20 frames (80 ms). Threshold parameters for particle detection were set to the default values of 1.5 (threshold 1) and 0.25 (threshold 2). For each dataset, a calibration of ratiometric contrast to mass was performed using a protein standard while using the same acquisition parameters, similar to a previously reported procedure60,67.
SLB preparation
SLBs were prepared using a similar procedure as previously reported, with small modifications21. In short, phospholipid stocks in chloroform were mixed to form a 5 mM stock solution with a molar composition of 0.05 mM DGS-NTA, 0.1 mM 18:1 PEG550 and 4.85 mM POPC. The stock solution was stored at −20 °C. Before use, 50 μl of the lipid stock solution was added to 200 μl of chloroform in a clean glass tube. The chloroform was evaporated by manually rotating the tube while applying a weak flow of nitrogen, followed by 1 h of evaporation under vacuum. Lipids were hydrated by adding 0.5 ml of buffer (20 mM HEPES pH 7.4, 150 mM KCl), followed by two cycles of 20-min incubation in a 40 °C water bath, mixing between each cycle. The sealed tube was left at ambient room temperature for at least 2 h or overnight. The hydrated lipids were tip-sonicated in a 1.5-ml Eppendorf tube using a 2-mm tip probe at 30% power and 1 s pulse duration separated by 3 s waiting time for a total of 10 min sonication time (Vibra-Cell, Sonics & Materials). During sonication, the tube was kept in ice water. The sonicated lipids were centrifuged at 21,130 × g for 30 min at 4 °C, before taking 0.4 ml of the supernatant. Cleaned coverslips were treated with oxygen plasma for 5 min at 40% power and 0.6 mbar oxygen pressure (Zepto plasma cleaner, Diener Electronic). Immediately after plasma cleaning, a silicon gasket (GBL103280, Grace Bio-Labs) was placed at the centre of the coverslip and 30 μl of buffer (20 mM Tris pH 7.8, 150 mM NaCl, 2 mM MgCl 2 ) followed by 20 μl of lipids were added and thoroughly mixed in the gasket and SLB formation was allowed for about 20 min. After examining the SLB integrity, excess vesicles were washed from the surface with assembly buffer.
Preparation of the histidine tag mi3-VLPs for measurements on SLB
Spytag-polyhistidine peptide (Spy-hist) at a concentration of 270 μM in DPBS was mixed with an 84-μM solution of mi3-VLP (total mi3-monomer concentration) at a volume ratio of 2:1, resulting in a large excess of the Spy-hist peptide (180 μM versus 27 μM) and the mixture was incubated on ice for 3 h. Following incubation, the solution was filtered through a 4-ml, 100-kDa MWCO centrifugal filter (Amicon) at 4,000 × g eight times to remove the excess peptide. For each round of centrifugation, the 4-ml initial solution was concentrated to 0.1 ml. This resulted in an estimated dilution factor for the excess of Spy-hist peptide of 109. To tether the subunits to the SLBs, the tagged VLPs were disassembled by diluting 1 μl of the tagged VLP solution into 50 μl of 2.5 M of guanidinium thiocyanate. After about an hour, the disassembled VLPs were rapidly diluted 100-fold into 20 mM HEPES pH 7.4, 138 mM NaCl (assembly buffer). The final concentration of tagged mi3-monomers is estimated to be 5 nM, which is much lower than the critical concentration for VLP formation. MP measurements validated the existence of only mi3-monomers and mi3-trimers in solution, before addition as a solution on top of the SLBs.
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