Source 15 rpc

Full-length murine SAA1.1 protein was recombinantly expressed with a pMal-c2X vector in Escherichia coli RV308 and purified as described previously^{26 (link)}.Fragments of mSAA have been generated by introduction of stop codons into the mSAA gene at specific positions using QuikChange XL Site-Directed Mutagenesis Kit (Stratagene). Expression of the fragments was performed in Escherichia coli Rosetta at 30 °C using ZYP-5052 autoinduction medium. Purification of the fragments has been done in seven steps: (i) amylose resin high flow (New England Biolabs) chromatography, (ii) nickel-Sepharose fast flow (GE Healthcare) chromatography, (iii) fusion protein cleavage by overnight incubation with tobacco etch virus protease at 34 °C, (iv) nickel-Sepharose fast flow chromatography to separate SAA from the fusion protein and maltose-binding protein, (v) Source 15 RPC (GE Healthcare) reversed-phase chromatography, (vi) size exclusion chromatography with a Superdex 75 (GE Healthcare) and (vii) Source 15 RPC (GE Healthcare) reversed-phase chromatography. The purified protein was lyophilized using an alpha 2–4 LDplus freeze dryer (Christ).

The human full-length α-synuclein and Gln79-α-synuclein proteins were recombinantly expressed following procedures described recently [57 (link)]. Purification included Ni²⁺-chelating chromatography on a Streamline Chelating resin (Streamline Chelating, GE Healthcare Life Sciences, Uppsala, Sweden). Fractions containing the expression construct were subjected to a second purification step via a glutathione sepharose resin (Glutathione Sepharose 4FF, GE Healthcare Life Sciences). The removal of glutathione was achieved by overnight dialysis against buffer containing 100 mM NaCl, 30 mM Tris/HCl pH 7.6, 0.1 mM DTT and a membrane with 6–8 kDa cutoff. Separation of the GST- and His-tag from the α-synuclein sequence by a TEV protease cleavage left an native N-terminus [53 (link)] followed by cyclization of Gln79-α-synuclein to pGlu79-α-synuclein with QC overnight at room temperature. The fractions obtained were analyzed and subjected to reversed phase chromatography (Source 15 RPC, GE Healthcare Life Sciences), followed by lyophilization and anion exchange chromatography (MonoQ 5/50GL, GE Healthcare Life Sciences). The final buffer used for the experiments was 20 mM Tris/HCl, pH 7.0, containing 100 mM NaCl. The purity of the samples was assessed by SDS PAGE and mass spectrometry. Protein concentrations were determined using UV absorption at 280 nm.

Protein solutions were first diluted with 50 mM ammonium bicarbonate (ABC) and reduced with 5 mM dithiothreitol (DTT, Merck) at 60°C for 45 min, followed by cysteine-blocking with 10 mM iodoacetamide (IAM, Sigma) at 25°C for 30 min. The samples were then diluted with 25 mM ABC and digested with sequencing-grade modified porcine trypsin (Promega) at 37°C for 16 hr. The peptides were desalted using a homemade C18 microcolumn (SOURCE 15RPC, GE Healthcare) and stored at –30°C until use.