Kta purifier 10 system

Delipidated vesicle and matrix pellets containing carbohydrates and proteins were washed twice with acetone, dried under a stream of nitrogen, and reconstituted in 3 ml of 20 mM bis-Tris/HCl (pH 6.5) loading buffer. Aliquots were chromatographically desalted on a HiPrep 26/10 Desalting column (GE Healthcare Life Sciences, Uppsala, Sweden) and then separated on an anion exchanger HiPrep 16/10 DEAE FF column (GE Healthcare Life Sciences) equilibrated with 20 mM bis-Tris/HCl (pH 6.5). Carbohydrate positive flow-through fractions were pooled together, lyophilized, resuspended in 15% acetonitrile in 150 mM ammonium bicarbonate, and applied to gel filtration on a HighPrep 16/60 Sephacryl S-300 HR column (GE Healthcare). All chromatographic separation steps were performed at room temperature on the high-performance liquid chromatography ÄKTA-Purifier 10 system (GE Healthcare Life Sciences).

The samples (200 µL) were separated by size on a fast protein liquid chromatography system (GE ÄKTA Purifier 10 System with Monitor UV-900 and Sample Pump P-900). The SEC was carried on a calibrated Superdex 75 Increase 10/300 GL column at room temperature with a flow of 0.5 mL/min PBS buffer at pH 7.5 over 1.5 column volumes (CVs). The column was equilibrated over 2 CVs (1 CV = 24 mL) of running buffer before the sample injection with monitoring at 215, 280, and 663 nm. The molecular weight of each sample was calculated using a linear calibration curve of partition coefficient K_av $K_{av}=\left(\frac{retentionvolume-Voidvolume}{Columnvolume-Voidvolume}\right)$
versus log(MW) generated by standard proteins; Conalbumin (75 kDa), Ovalbumin (43 kDa), Carbonic Anhydrase (29 kDa), Ribonuclear (13.7 kDa), and Aprotinin (6.5 kDa) (Fig. 1). The Stoke’s radius (R_s) of each sample was calculated using a linear calibration curve of $\sqrt{-\log \left(K_{av}\right)}$ versus R_s of standard proteins; Conalbumin (36.4 Å), Ovalbumin (30.5 Å), Carbonic Anhydrase (23 Å), Ribonuclear (16.4 Å), and Aprotinin (13.5 Å) (Supplementary Fig. 22d).

Human sera were obtained from healthy volunteers aged between 30 and 50 years. Healthy donors were recruited at the University of Heidelberg (Germany), after giving their written informed consent. The protein G purified serum IgG was obtained from a clinical study approved by the appropriate local Ethics Committee and Institutional Review Board of the University of Heidelberg (S-040). 100 µL-aliquots were diluted with 900 µL PBS and applied on a HiTrap Protein G HP column (1 mL) using an ÄKTA purifier 10 system (GE Healthcare, Munich Germany) at 4 °C and a flow rate of 0.5 mL/min. After washing with 10 mL PBS, pure IgG fractions were eluted with 100 mM glycine pH 2.6 and immediately neutralized with 1.5 mM Tris-HCl (pH 8.8). Purity of the obtained samples was checked by SDS electrophoresis and Coomassie staining.

IolR-His₆ was overproduced from the plasmid pET28b-IolR in E. coli BL21 (DE3) and S. Typhimurium strain 140282 (link). Overnight culture of the strains were diluted 1:100 into 400 ml of LB medium supplemented with 50 μg/ml kanamycin and incubated for 3 h at 37 °C with rotation at 180 rpm. At an OD₆₀₀ of 0.6, IolR production was induced by adding 0.1 mM isopropyl-β-_D-1-thiogalactopyranoside (IPTG). After an overnight incubation at 37 °C and 180 rpm, the cells were harvested by centrifugation at 4 °C and 7500 rpm for 20 min. The pellets were each resuspended in 5 ml of native lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl and 10 mM imidazole at pH 8.0) and lysed by three passages through a French press (SLM Aminca Instruments, Rochester, NY, USA) at 900 psi; residual cell debris was removed twice by centrifugation at 4 °C and 9000 rpm for 15 min. Following filtration, IolR was isolated using a HisTrap HP colunm with an ÄKTA purifier 10 system (GE Healthcare, Little Chalfont, UK). The protein concentration was determined using RotiQuant solution (Carl, Roth GmbH, Karlsruhe, Germany) according to the Bradford method32 (link). The purity of the eluted fractions was analysed by separation on a 15% sodium dodecyl sulfate (SDS)-PAA gel.

The native molecular mass of the purified proteins was determined by gel filtration chromatography using a Superdex 200 10/300 GL column (GE Healthcare, China) according to the manufacturer’s instructions. The column was pre-equilibrated and eluted with Tris-HCl buffer (50 mM, pH 7.5) containing 0.15 M NaCl at a flow rate of 0.5 mL min⁻¹, which was controlled by an ÄKTA Purifier 10 system (GE Healthcare, China). The standard proteins used for calculating the native molecular mass of enzymes were carbonic anhydrase (30 kDa), bovine serum albumin (67 kDa), alcohol dehydrogenase (150 kDa), catalase (200 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa).

Bacteria from expression cultures were suspended in standard binding buffer for Ni-affinity chromatography and lysed by sonification. After clearing of the lysate, the proteins were purified by Ni-affinity chromatography using an ÄKTA purifier 10 system (GE Healthcare, Uppsala, Sweden). Cleavage of the N-terminal His tag was done by using TEV protease (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s recommendations. Additional purification steps via cation- or anion-exchange chromatography or size exclusion chromatography were performed if necessary. The purity of the recombinant expressed proteins was tested by Coomassie blue-stained SDS-PAGE.
HEK293-derived Fn derivates were purified from the supernatants by gelatin agarose affinity chromatography method as described elsewhere (28 (link)). The success of the purification was proven by dot immunoassay using monoclonal anti-GFP antibody (Abcam, Cambridge, MA).