Desalting column

Total enzyme extraction and VAcInv activity assays were conducted at 4°C as previously described [34 (link)]. The extraction buffer contained 50 mM HEPES (pH 7.5), 15 mM MgCl₂, 2 mM EDTA, 2 mM dithiothreitol (DTT), 10% v/v glycerol and 2% PVPP, 2.0 μg mL^-1 leupeptin, and 1 mM PMSF. Supernatant was desalted with 2 mL desalting column (ThermoFisher Scientific, NY, USA). desalting columns were pre-equilibrated with desalting buffer containing 50 mM HEPES (pH 7.5), 15 mM MgCl₂, 2 mM EDTA, 2 mM DTT, and 2.0 μg mL^-1 leupeptin. Desalted extracts were stored at -80°C freezer until assay.

According to the instruction of the EZ-Link Desthiobiotinylation and Pull-Down Kit (Thermo Fisher Scientific, Cat# 16138), purified human FH (Merck, Cat# 341274) was desthiobiotin-labeled using 15-fold molar excess EZ-Link sulfo-NHS-LC-desthiobiotin solution. Excess desthiobiotin reagents were removed using a desalting column (Thermo Fisher Scientific, Cat# 89891). Then, the desthiobiotin-FH samples were incubated with streptavidin agarose resins at 4°C for 2 h. Desthiobiotin-glycine was used as a control. The agarose resin was washed thrice with PBS and incubated with the ExPEC membrane protein solution at 4°C for 4 h. After thorough washing, the bound membrane proteins were eluted with the biotin elution buffer.

The HSA-PEG-Maleimide (HSA-PEG-Mal) polymer was self-assembled using a method previously described in the literature [17 (link)]. To summarize, HSA-PEG-Mal was dissolved in dimethyl sulfoxide and gradually added to deionized water. The resulting solution underwent dialysis against deionized water for 24 h and was subsequently centrifuged. The supernatant was collected and stored at 4 °C. To thiolate collagenase I, the Traut reaction was employed by introducing 5 × Traut to the collagenase I solution (5 mg/mL in phosphate-buffered saline [PBS]) and stirring the mixture at 25 °C for 1 h. The obtained product, namely thiocollagenase I (SH-Collagenase I), underwent purification using a desalting column (Thermo Fisher Scientific, Waltham, MA, USA). Next, HSA-PEG-Mal was reacted with SH-collagenase I at 25 °C overnight to prepare pre-C-HSA. Then, Mal-PEG-COOH was added and reacted at room temperature for 4 h. SH-PEG-COOH was added to the solution and reacted at 25 °C for 4 h. The HSA-C solution was obtained and centrifuged for 30 min. The supernatant was removed, and the pelleted polymer was resuspended in PBS and stored at 4 °C. Using a Zetasizer Nano (Malvern Instruments, Malvern, UK) to characterize the hydrated particle size distribution and zeta potential.