Carbonic anhydrase

First, size-exclusion chromatography was applied to determine the oligomerization of purified OmpAVac as previously described (Gao et al., 2017 (link)a), with slight modifications. The standard proteins Blue dextran 2000, aldolase, conalbumin, ovalbumin, carbonic anhydrase (CA), and ribonuclease A (R) were purchased from GE Healthcare. OmpAVac and the protein standards were diluted to 10 mg/mL and loaded onto a Superdex 75/300 column in buffer (20 mM Tris, pH 8.0). The elution peaks of OmpAVac and the protein standards were recorded. The predicted Mw of OmpAVac was calculated as described by Haiguang et al. (Wang et al., 2016 (link)). For the dynamic light-scattering assay, purified OmpAVac was diluted to 0.5 mg/mL and loaded onto a Zetasizer (Malvern, UK) equipped with an argon ion laser. The analysis was then performed three times at 25°C.
The cross-linking reaction of OmpAVac was performed using glutaraldehyde as a linking agent (Fadouloglou et al., 2008 (link)). In brief, 30 ng/mL OmpAVac was incubated with glutaraldehyde at 4°C for 10 h. The final concentration of glutaraldehyde in each reaction was adjusted to 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5. The reaction was stopped by the addition of SDS loading buffer. Finally, the protein samples were visualized via SDS-PAGE.

We performed gel filtration experiments for bOBP and its mutant forms in a buffered solution without and with addition of GdnHCl using a Superdex-75 PC 3.2/30 column (GE Healthcare, Sweden) and an AKTApurifier system (GE Healthcare, Uppsala, Sweden). The column was equilibrated with the buffered solution or GdnHCl at the desired concentration, and 10 µl of the protein solution prepared under the same conditions was loaded on the pre-equilibrated column. The change in hydrodynamic dimensions for the studied proteins was evaluated as a change in the bOBP or the mutant protein elution volume. Multiple proteins with known molecular masses (aprotinin (6.5 kDa), ribonuclease (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa) and conalbumin (75 kDa), which are chromatography standards from GE Healthcare) were used to generate the calibration curve.

The gel filtration with the FPLC system can be also employed to analyze the polymerization state of native or active proteins^{26 (link)}. Protein samples were injected into the column for separation, which was equilibrated with the buffer (20 mM Tris–HCl, 150 mM NaCl, 10% glycerol, pH 7.5). Five molecular weight markers were used: beta-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa) (GE Healthcare, China agency). Protein separation was monitored by measuring the absorbance at 280 nm. To analyze the effects of acidic or more alkaline conditions on the protein polymerization state, the above-mentioned buffer was adjusted to pH 5.5 and 8.5, respectively.