Stirred ultrafiltration cell

Enzyme productions were carried out from one selected recombinant clone expressing parental or chimeric enzymes. Recombinant P. pastoris were cultivated at 30 °C in 400 mL of BMGY (Buffered Glycerol-complex Medium) during 16 h until reaching ~4 A₆₀₀, concentrated in 100 mL of BMMY (0.5% methanol) and then cultivated for 72 h with regular methanol addition every 24 h. Culture supernatants were collected by centrifugation (3000 g for 15 min) and recombinant enzymes in supernatant were concentrated to 20 mL on 3 kDa ultrafiltration membrane using Stirred Ultrafiltration Cell (Millipore®). Parental and chimeric His-tagged enzymes were then purified by Ion Metal Chromatography Affinity (IMAC) (Protino® Ni-NTA, Macherey-Nagel®), and analyzed by SDS-PAGE (12%, wt/vol.) for the validation of proteins integrity and purity. Protein concentrations were determined by Bradford method using bovine serum albumin as standard.

Pure water permeability measurements were performed using a Millipore stirred ultrafiltration cell (model 8050). Each membrane was placed in the ultrafiltration cell, and a transmembrane pressure of 2 bar was applied using an air gas cylinder. Water flowed through the membrane for 30 s. Permeability was calculated as the quotient of the mass of water permeated and the multiplication product of the surface area, flow time, and transmembrane pressure. Experiments were repeated in triplicate. Reported values are the averages, with error bars representing the standard deviation.

The murine
H-2D^b heavy chain and mouse β2m (mβ2m) were
expressed individually as inclusion bodies using BL21 (DE3) E. coli strain, following previously published protocols.^{71 (link)−74 (link)} gp33, V3P, F6f, and V3P_F6f peptides were synthetized and used to
refold pMHC, yielding H-2D^b/gp33, H-2D^b/V3P,
H-2D^b/F6f, and H-2D^b/V3P_F6f, respectively.
H-2D^b/peptide complexes were obtained through dilution.^{75 (link)} Briefly, peptide (10 μM) and mβ2m
(2 μM) were incubated in the refolding buffer (100 mM Tris pH
8, 450 mM l-arginine, 5 mM l-glutathione reduced,
0.5 mM l-glutathione oxidized, 2 mM EDTA, and 0.5 mM AEBSF)
at 4 °C under stirring for 30 min. The unfolded H-2D^b heavy chain, solubilized in 6 M guanidinium hydrochloride, was thereafter
added to a final concentration of 1 μM. The refolding was completed
after 72 h at 4 °C under stirring. The solution was concentrated
using Stirred Ultrafiltration Cell (Millipore) and Amicon Ultra-15
Centrifugal Filters (EMD Millipore). Finally, all pMHC complexes were
purified by size exclusion chromatography using a HiLoad 16/600 Superdex
200 pg column (GE Healthcare), preliminarily equilibrated in 20 mM
Tris–HCl pH 7.4.

Water flux was measured as was described in previous work [48 (link)] using a Millipore stirred ultrafiltration cell with 13.4 cm² active transport area. Dead-end operation mode was used.

Butyrivibrio fibrisolvens strain DSMZ 3071 (B. fibrisolvens 3071) was obtained from the DSMZ culture collection. B. fibrisolvens 3071 was cultivated to the late stationary phase under anaerobic conditions at 37 °C in medium M10 [26 (link)] without rumen fluid. Various substrates including 4% (w/v) D-xylose (Sigma-Aldrich), 4% (w/v), D-glucose (Sigma-Aldrich), 4% (w/v) beechwood xylan (Fluka), and the combination of 3% (w/v) beechwood xylan (Fluka) with 1% (w/v) D-glucose (Sigma-Aldrich) were used as carbon sources. Bacteria were collected by centrifugation at 10000 g for 20 min at 4 °C. The supernatants containing extracellular enzymes were further processed at 10 °C in a stirred ultrafiltration cell (Millipore) using a Millipore PES membrane with a 10 kDa cut off and stored immediately at − 24 °C. The 6-fold concentrated extracellular enzyme extracts were used for monitoring the proteins secreted in response to the different growth substrates.

The pET3a-His-DJ1 plasmid was a kind gift from Michael J Fox Foundation MJFF (Addgene plasmid # 51488). The His₆-tagged DJ-1 C106A mutation was cloned by inverse PCR using pET3a-His-DJ1 as the template and the following primer sequences: 5′-GCCGCCATTGCCGCAGGCCCGACCGC-3′ and 5′-AATCAGGCCTTTGCGGTTCTCCTGCTCTTTCAGG-3′. The His₆-tagged wild-type DJ-1 and DJ-1 C106A mutation were expressed in E. coli Rosetta (DE3) cells with an overnight IPTG induction at 16 °C. The bacterial pellet was lysed by sonication and lysate cleared by centrifugation at 12,000 r.p.m. for 30 min. Lysate was loaded on Ni²⁺-NTA agarose (GE Healthcare) affinity column and eluted on AKTA FPLC, followed by desalting using Zeba Spin Desalting Columns (7 K MWCO, 10 mL) according to the manufacturer’s protocol. Purified recombinant proteins were analyzed by SDS-PAGE, and concentrated using stirred ultrafiltration cells (Millipore) according to the manufacturer’s protocol. The concentration of each protein was determined using 280 nm wavelength on a NanoDrop 2000c (Thermo Scientific).