Vivaspin 4

We synthesized N-terminal His-tagged YGFP derivatives, GFPuv and EGFP by standard PCR techniques and cloned them into the pEGFP vector at the Hind III and EcoR I sites, as described above. Protein expression was analyzed in DH5α cells at 37°C without any induction. After overnight incubation, cells were resuspended in PBS containing cOmplete^™ Mini EDTA-free protease-inhibitor cocktail (Roche, Basel, Switzerland) and sonicated for 3 min (30 sec x 6 pulses) on ice with Q500 (Qsonica, LLC. Newton, CT, US). The soluble protein fraction was mixed with TALON Superflow Metal Affinity Resin (Takara Bio Inc) and incubated overnight at 4°C. After incubation, the resin was washed 5 times with PBS and then washed in PBS containing 200 mM imidazole to elute His-tagged proteins. Eluted proteins were subjected to VIVASPIN4 (Sartorius Stedim, Göttingen, Germany) centrifugal concentration for buffer exchange. To determine protein concentration, purified proteins dissolved in PBS were diluted 2-fold with 10% SDS and then incubated at 95°C for 10 min. Protein concentration of the quenched sample was determined using a BCA protein assay kit (Pierce Biotechnology, Rockland, IL, USA).

For clinical proteomics, 10 ml serum samples were taken from RA patients at visits V1, V5 and V6. Sera were then subjected to high-performance liquid chromatography-based immunodepletion for high abundant proteins using Human-14® immunoaffinity columns (Agilent, Boeblingen, Germany). The depletion of high abundant proteins from sera was carried out according to the manufacturer’s protocol. Subsequently, 6 M urea was added and the samples containing the low abundant protein fractions were concentrated using Vivaspin 4® (Sartorius Stedim, Goettingen, Germany) ultrafiltration spin columns. Samples were mixed with ice-cold precipitation solution (20% trichlororacetic acid in acetone) at a ratio of 1:3. After centrifugation the protein pellets were washed with ice-cold acetone and dried for 5 minutes. The total protein concentration was estimated using the Bradford method with bovine serum albumin (1 mg/ml) as the standard [18 (link)] using the LAMBDA™ 25 spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA).

In order to characterize the isolated M. melolontha β-glucosidase genes, they were heterologously expressed in a line of T. ni-derived cells (High Five Cells; Life Technologies, Carlsbad, CA, USA) as described in Rahfeld et al., 2015 (link). Briefly, genes were cloned into the pIB/V5-His TOPO vector (Life Technologies). After sequence verification, these vector constructs were individually used with the FuGeneHD-Kit to transfect insect High Five Cells according to the manufacturer’s instructions (Promega, Madison, WI, USA). After 1 day of incubation at 27°C, the cultures were supplied with 60 mg*ml^–1 blasticidin (Life Technologies) to initiate the selection of stable cell lines. Afterwards, the insect cells were selected over three passages. The cultivation of the stable cell lines for protein expression was carried out in 75 cm³ cell culture flasks, containing 10 ml Express Five culture medium (Life Technologies), 20 mg*ml^–1 blasticidin, and one x Protease Inhibitor HP Mix (SERVA Electrophoresis, Heidelberg, Germany). After 3 days of growth, the supernatant was collected by centrifugation (4000 ×g, 10 min, 4°C), concentrated using 10.000 Vivaspin 4 (Sartorius), and desalted (NAP-5; GE Healthcare, Munich, Germany) into assay buffer (100 mM NaPi, pH 8).