2.4 VLP-GOx nanoreactors synthesis
The VLP-GOx nanoreactors were synthesized by combining the disassembled BMV capsid and free-GOx enzyme and using the auto-assembly method. First, the disassembled CP protein was mixed with the free-GOx enzyme (in phosphate buffer pH 7.0) at different molar ratios (1:2, 1:3, 1:6, and 1:23 GOx:CP). Then, the GOx:CP mixtures were dialyzed in two steps. First, initial buffer (50 mM NaCl, 10 mM KCl, 5 mM MgCl2, 50 mM Tris, pH 7.2); and second, auto-assembly buffer (50 mM sodium acetate, 750 mM NaCl, 8 mM magnesium acetate, pH 5.1), 4 h, 4°C. After dialysis, the samples were centrifuged at 40,000 rpm, 4°C for 2 h to obtain the nanoreactors in a pellet. After centrifugation, the supernatant was removed and the pellet containing the VLP-GOx nanoreactors was resuspended in the auto-assembly buffer. The resulting VLP-GOx nanoreactor suspensions were filtered using a 0.2 µm filter for further high-performance liquid chromatography (HPLC) purification. Next, the nanoreactors suspension was injected in a size exclusion column (TSKgel Column, Super SW2000, Supelco-Tosoh Bioscience, Bellefont, PA) for HPLC (Agilent 1100 series) purification of the VLP-GOx nanoreactors. The VLP-GOx nanoreactor fractions were collected and then ultrafiltered through a 30,000 Da membrane (Merck Amicon™ Ultra-15 Centrifugal Filter Units) to adjust the work concentration. Finally, the resulting VLP-GOx nanoreactors were estimated by Dynamic Light Scattering (DLS) with a Zetasizer Nanoseries device (Nano-ZS, Malvern Instruments). The total amount of protein in all preparations was estimated via the Bio-Rad Bradford Protein Assay (Hercules, CA).