Kta purifier

The molecular masses of the purified proteins in a denatured state were analyzed by SDS-PAGE (53 (link), 54 ). Immunodetection of the His₁₀-tagged proteins was performed via Western blotting (55 (link)) using a nitrocellulose blotting membrane (Amersham Protran 0.45-μm NC, GE Healthcare, Germany) and a Penta-His horseradish peroxidase (HRP) conjugate antibody (Qiagen, Germany).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min⁻¹ with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M⁻¹cm⁻¹ (StyI) and 48,150 M⁻¹cm⁻¹ (StyJ) as well as molecular weights of 29,836.3 g mol⁻¹ (StyI) and 30,095.6 g mol⁻¹ (StyJ) as predicted by Expasy ProtParam (56 (link)).

The oxidized RNA was dissolved in 250 µL water containing 20 mM imidazole (pH 8), 5 mM NaCNBH₃, 1 mM EDTA and 1 mM cytidine derivative 1. The reaction was carried out at 37 °C. After 2 hours, 25 µL of 50 mM NaBH₄ were added, and the reaction mixture was incubated for additional 15 min. Ethanol precipitation yielded the crude coupling product, which was analyzed by reversed-phase HPLC on an Äkta Purifier (Amersham Bioscience). Column: Macherey Nagel EC 250/4 Nucleodur 100-5 C18 ec; Buffers: (A) 0.1 M triethylammonium acetate (pH 7.0), 5% acetonitrile and (B) 0.1 M triethylammonium acetate (pH 7.0), 30% acetonitrile; flow rate 0.5 mL min⁻¹; gradient: 0% → 85% (B) in 14 CV, 85% 4 CV, 85% → 100% (B), 100% (B) 4 CV, 100% → 0% (B) 2 CV. The product containing fraction was collected, RNA was lyophilized and analyzed by MALDI–MS: (calcd mass: 7339.9 g/mol, found: 7339.04 [M + 1]⁺; 3668.515 [M + 2]⁺/2, Supporting Information File 1).

The molecular masses of the purified proteins in a denatured state were analyzed by SDS-PAGE (53 (link), 54 ). Immunodetection of the His₁₀-tagged proteins was performed via Western blotting (55 (link)) using a nitrocellulose blotting membrane (Amersham Protran 0.45-μm NC, GE Healthcare, Germany) and a Penta-His horseradish peroxidase (HRP) conjugate antibody (Qiagen, Germany).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min⁻¹ with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M⁻¹cm⁻¹ (StyI) and 48,150 M⁻¹cm⁻¹ (StyJ) as well as molecular weights of 29,836.3 g mol⁻¹ (StyI) and 30,095.6 g mol⁻¹ (StyJ) as predicted by Expasy ProtParam (56 (link)).

rAAV1 were produced by cotransfection of pTR-UF11³ and pDP1-rs (Plasmid Factory—no. PF401) with PEI (Polyethylenimine—PolyScience—no. 23966-1) in adherent HEK-293 with DMEM (Gibco—no. 41966-029) supplemented with 10% FBS (Pan Biotech—no. P30-3031). Briefly, 72 h post-transfection, adherent cells were detached by addition of 12.5 mM final concentration of ethylenediaminetetraacetic acid (EDTA) 0.5 M. Supernatant and cells were treated with 30 mM MgCl₂, 0.5% Triton X-100 and 1 000 U (for 500 mL of bulk) of Benzonase (Roche—no. 1.01654-0001) for 3 h at 37°C under 140 rpm (orbital shaker—Heathrow Scientist).
Culture bulk was then clarified with a Pall Preflow filter (no. DFA3001UBC). rAAV1 were purified from the clarified product with Poros Go-Pure AAVX prepacked column (Thermo Fisher—no. A36652) and an Äkta purifier (Cytiva). rAAV1 were eluted with 100 mM Glycine buffer at pH 2.5 and neutralized with Tris pH 8.5. rAAV1 were concentrated with PBS supplemented with 625 mM KCl and 10 mM MgCl₂ with an Amicon 100 kDa (Millipore—no. UFC910024).

Membranes purified from yeast expressing His₈-GFP-TEV-FICD were diluted to 2 mg/mL in Lysis Buffer 1 and supplemented with a detergent (see Table 1) at a final concentration of 2, 4 or 6 mg/mL. When indicated, CHS was included to one third of the detergent concentration. The mixtures were incubated at 4 °C for 1 h with gentle agitation. Insoluble material was collected by centrifugation at 160,000× g for 30 min at 4 °C. The solubilisation efficiency was estimated by measuring the GFP fluorescence (Fluoroskan Ascent, Thermo Fisher Scientific, Waltham, MA, USA; excitation at 485 nm and emission at 520 nm) in the soluble fraction before and after the centrifugation [32 (link)]. For FSEC, 350 μL of the solubilised membrane proteins were separated on a Superose 6 Increase 10/300 GL (Cytiva, Marlborough, MA, USA) column connected to an ÄKTA purifier (Cytiva, Marlborough, MA, USA) in SEC Buffer 1 (25 mM Tris-HCl pH 7.6, 500 mM NaCl, 10% glycerol, 0.3 mg/mL DDM). An in-line fluorescence detector (Shimadzu RF-20A prominence, Shimadzu Corporation, Kyoto, Japan) was used to record the elution of the GFP-tagged FICD. All detergents used for detergent screening were purchased from Affymetrix (Santa Clara, CA, USA) or GlyconBiochemicals (Luckenwalde, Germany) and are listed in Table 1.

A total of 250 μl of purified TPR-like domain at 1–3 mg/ml were fractionated by SEC using a Superdex 200 10/300 column (Cytiva) equilibrated in 20 mM Tris-HCl pH 6.8, 0.2 M NaCl, 5% glycerol, and 1 mM DTT and an ÄKTA purifier (Cytiva) at a flow rate of 0.5 ml/min. The eluted samples were characterized by measuring the refractive index and multi-angle light scattering (MALS), using Optilab T-rEX and DAWN 8⁺ (Wyatt). Data were analyzed using the Astra6 software (Wyatt) to obtain the molar mass of each protein and represented using GraphPad Prism 9.