Purification of the 9‐hcLAAO4 fusion protein was carried out at 4°C. After the removal of the cells by centrifugation, the supernatant with the secreted 9‐hcLAAO4 in the medium was loaded onto Ni2+‐NTA resin. The flow‐through was caught, mixed, and reloaded onto Ni2+‐NTA resin via a peristaltic pump for 24 hr and a flow velocity of 1 ml/min. The column was washed with 40 column volumes (cv) of ‐washing buffer (50 mM Na2HPO4 pH 7.0, 300 mM NaCl, and 20 mM imidazole). After washing, 9‐hcLAAO4 was eluted with His buffer (50 mM Na2HPO4 pH 7.0, 300 mM NaCl) containing different concentrations of imidazole (50, 100, 250, and 500 mM). The enzyme was concentrated via ultrafiltration (Vivaspin 6 30.000 MWCO, Sartorius), rebuffered into HEPES buffer pH 7.0 (100 mM HEPES, 150 mM NaCl) and stored at 4°C. Size exclusion chromatography was performed according to Bloess et al., 2019 with an Ettan LC (GE Healthcare Life Sciences, Chicago, IL, USA) on a Superdex® 200 Increase 10/300 GL column (GE Healthcare Life Sciences, Chicago, IL, USA).
Vivaspin 6 30.000 mwco
Manufactured by Sartorius
The Vivaspin 6 30.000 MWCO is a centrifugal concentrator device used for the concentration and buffer exchange of macromolecular solutions. It features a 30,000 molecular weight cut-off (MWCO) membrane, which allows the separation of molecules based on their size.
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2 protocols using vivaspin 6 30.000 mwco
1
Purification of 9-hcLAAO4 Fusion Protein
2
Purification of Recombinant L-Amino Acid Oxidase
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Purification was performed at 4°C as described (Heß et al., 2020 (link)). The supernatant with the secreted LAAO was loaded onto a Ni2+‐NTA resin. The flow‐through was collected, mixed, and reloaded onto the Ni2+‐NTA resin via a peristaltic pump for 24 h with a flow velocity of 1 ml min−1. The column was washed with 10 column volumes (cv) of His‐washing buffer (50 mM Na2HPO4 pH 7.0, 300 mM NaCl and 20 mM imidazole). LAAO was eluted with 10 ml His buffer (50 mM Na2HPO4 pH 7.0, 300 mM NaCl and 500 mM imidazole). The enzyme was concentrated via ultrafiltration (Vivaspin 6 30.000 MWCO, Sartorius), rebuffered into HEPES buffer pH 7.0 (100 mM HEPES, 150 mM NaCl), and stored at 4°C. Size exclusion chromatography was performed according to Bloess et al., 2019 (link) with an Ettan LC (GE Healthcare Life Sciences, Chicago, IL, USA) on a Superdex® 200 Increase 10/300 GL column (GE Healthcare Life Sciences, Chicago, IL, USA).
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