title: A method of purifying intact complement factor H from human plasma.
abstract: The aim of this study was to establish a method of purifying intact complement factor H (CFH) from human plasma. CFH was isolated from human plasma by polyethylene glycol (PEG) precipitation, following three sequential chromatographic columns, which consisted of l-lysine Sepharose column, Resource Q column and Sephacryl S-300 High Resolution HiPrep 16/60 column. All the above steps were performed at 4°C by Fast Protein Liquid Chromatography (FPLC) AKTA Purifier 10 with Frac-900. Identification of the purified CFH was confirmed by SDS-PAGE and Western blot. The following functions of the purified CFH were further analyzed compared with the commercial CFH in vitro: (1) binding ability with C3b; (2) binding ability with mCRP; (3) the protecting function of the hemolysis of sheep red blood cells; (4) the cofactor role for complement factor I-mediated proteolytic inactivation of C3b. Homogeneous CFH was purified from the plasma fraction through the above four steps. The purity and the functions of the purified CFH were comparable to the commercial CFH. The yield of CFH was 26±3% in our study. Compared with previous methods, our method was high yield with high purity. We established a stable and feasible system for purifying intact CFH, which could be used in the lab and clinical investigations.
authors.affiliation: Renal Division, Department of Medicine, Peking University First Hospital, Beijing 10034, China; Institute of Nephrology, Peking University, Beijing 10034, China; Key Laboratory of Renal Disease, Ministry of Health of China, Beijing 10034, China; Key Laboratory of Chronic Kidney Disease Prevention and Treatment, Ministry of Education of China, Beijing 100034, China.
authors.forename: Feng-Mei
authors.initials: FM
authors.lastname: Wang
authors.forename: Feng
authors.initials: F
authors.lastname: Yu
authors.forename: Ming-Hui
authors.initials: MH
authors.lastname: Zhao
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002097:C-Reactive Protein / Q000737:chemistry / Q000378:metabolism; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003179:Complement C3b / Q000737:chemistry / Q000378:metabolism; D017242:Complement Factor H / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D011092:Polyethylene Glycols / Q000737:chemistry; D012756:Sheep
chemical_list: D011092:Polyethylene Glycols; D003179:Complement C3b; D017242:Complement Factor H; D002097:C-Reactive Protein
keywords: Chromatography; Complement factor H; Human plasma; Purification