New process for purifying high purity α1-antitrypsin from Cohn Fraction IV by chromatography: A promising method for the better utilization of plasma.
α1-antitrypsin (AAT) is a 52kDa serine protease inhibitor that is abundant in plasma. It is synthesized mainly by hepatic cells, and widely used to treat patients with emphysema due to congenital deficiency of AAT. A new isolation method for the purification of AAT from Cohn Fraction IV (Cohn F IV) is described. Cohn F IV is usually discarded as a byproduct from Cohn process. Using Cohn F IV as starting material does not interfere with the production of other plasma proteins and the cost of purification could be reduced greatly. Parameters of each step during purification were optimized, 15% polyethyleneglycol (PEG) concentration and pH 5.2 for PEG precipitation, elution with 0.05M sodium acetate and pH 4.7 for ion-exchange chromatography, and two steps blue sepharose affinity chromatography were chosen for AAT purification. The final protein with purity of 98.17%, specific activity of 3893.29 IU/mg, and yield of 28.35%, was achieved. Western blotting was applied for qualitative identification of final product, which specifically reacted with goat anti-human AAT antibody. LC-ESI-MS/MS was also employed to confirm the final protein. High performance liquid chromatography was used to analyze the composition of purified protein suggesting that pure protein was achieved. The molecular weight of AAT is 51062.77Da which was identified by LC-MS-MS. The manufacturing process described here may make better use of human plasma with Cohn F IV as starting material. The simple process described in this study is simple and inexpensive, it has a potential value for large scale production.
Beijing Key Laboratory of Blood Safety and Supply Technologies, Beijing Institute of Transfusion Medicine, Beijing 100850, China.
Chaoji
C
Huangfu
Beijing Key Laboratory of Blood Safety and Supply Technologies, Beijing Institute of Transfusion Medicine, Beijing 100850, China.
Jinchao
J
Zhang
Beijing Key Laboratory of Blood Safety and Supply Technologies, Beijing Institute of Transfusion Medicine, Beijing 100850, China.
Yuyuan
Y
Ma
Beijing Key Laboratory of Blood Safety and Supply Technologies, Beijing Institute of Transfusion Medicine, Beijing 100850, China.
Junting
J
Jia
Beijing Key Laboratory of Blood Safety and Supply Technologies, Beijing Institute of Transfusion Medicine, Beijing 100850, China.
Maomin
M
Lv
Beijing Key Laboratory of Blood Safety and Supply Technologies, Beijing Institute of Transfusion Medicine, Beijing 100850, China. Electronic address: zhaoxiong1980@126.com.
Xiong
X
Zhao
Beijing Key Laboratory of Blood Safety and Supply Technologies, Beijing Institute of Transfusion Medicine, Beijing 100850, China. Electronic address: zhangjg@bmi.ac.cn.
Jingang
J
Zhang
D001798:Blood Proteins / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006801:Humans; D008970:Molecular Weight; D015203:Reproducibility of Results; D000515:alpha 1-Antitrypsin / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*
D001798:Blood Proteins; C032309:Cohn fraction IV; D000515:alpha 1-Antitrypsin
Blue sepharose affinity chromatography; Cohn Fraction IV; High performance liquid chromatography; Ion-exchange chromatography; α1-antitrypsin