PMC:6093860 / 16742-18820 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/6093860","sourcedb":"PMC","sourceid":"6093860","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6093860","text":"Recombinant C9 purification\nHuman C9 and mouse C9 protein were purified using similar methods (with minor variations). The human C9 gene (P02748) was cloned into pSectag2a (Thermo Fisher Scientific) for expression in mammalian Expi293 cells where the native secretion sequence was replaced with the Igκ leader sequence. Human C9 mutants (F262C, V405C and F262C/V405C) were cloned using QuikChange. The mouse C9 (P06683) sequence was synthesised and cloned into pcDNA3.1 vector (GeneScript) also containing an Igκ leader sequence. Recombinant protein was produced by transient expression in Expi293F cells (Thermo Fisher Scientific) for four days according to the manufacturer’s instructions. The oligonucleotide primers used for cloning can be found in Supplementary Table 3.\nThe purification methods were essentially the same as previous one13,23 with some exceptions. Following centrifugation, the Expi293 media containing C9 was diluted with an equal volume of 10 mM sodium phosphate pH 7.4, 20 mM NaCl containing cOmplete protease inhibitor tablet (Roche). Then, it was loaded onto an equilibrated, HiTrap DEAE sepharose column (1 mL resin per 100 mL media). Chromatography steps were performed on an ÄKTA FPLC. The protein was eluted from the DEAE column using a linear gradient over 20 column volumes (from 10 mM sodium phosphate, 45 mM NaCl, pH 7.4 to 10 mM sodium phosphate, 500 mM NaCl, pH 7.4). Pooled fractions containing C9 were further purified using hydroxyapatite specifically using a pre-packed Bio-Rad type I CHT column equilibrated in 10 mM sodium phosphate pH 7.0, 100 mM NaCl. The CHT elution was performed over a six column volumes phosphate gradient at pH 8.1 (from 45 mM to 350 mM). Pooled fractions were concentrated using a 30 kDa MWCO concentrator (Amicon) and further purified using a size exclusion column; prepacked Superdex S200 16 mm × 60 mm or 26 mm × 60 mm (GE Healthcare life sciences). Size exclusion chromatography for human C9 was performed in 10 mM HEPES pH 7.2, 200 mM NaCl whereas mouse C9 was purified in 10 mM HEPES pH 7.2, 100 mM NaCl.","divisions":[{"label":"Title","span":{"begin":0,"end":27}}],"tracks":[]}