title: An improved method to extract and purify cystatin from hen egg white.
abstract: Hen egg white cystatin, an inhibitor of cysteine proteinase, may have wide applications for improving human health. However, its pricy cost associated with extraction and preparation has hurdled its further utilization. The objective was to develop an improved method to extract and purify cystatin from egg white. After removal of ovomucin, a fraction containing cystatin was obtained by cation exchange chromatography, and further purified by affinity chromatography using a cm-papain-Sepharose column. The prepared cystatin was then characterized by SDS-PAGE, Western-Blot, and LC-MS/MS, and its purity was determined by HPLC method instead of the conventional immunodiffusion method. The protein content of cystatin extract was 66.4 ± 2.3%. In comparison with the conventional method, the purity of cystatin was improved from 56.6 ± 1.7% to 93.3 ± 4.0%, and its yield was improved from 21.3 ± 1.2% to 33.6 ± 1.5%. Relative activities of cystatin to inhibit papain prepared by our method and the conventional method were determined to be 88 ± 7% and 91 ± 4% respectively, against a cystatin standard from Sigma. This suggested no significant loss of activity during the separation process.
authors.affiliation: Department of Agricultural, Food and Nutritional Science (AFNS), 4-10 Ag/For Building, University of Alberta, Edmonton, Alberta, Canada T6G 2P5.
authors.forename: Jiapei
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Agricultural, Food and Nutritional Science (AFNS), 4-10 Ag/For Building, University of Alberta, Edmonton, Alberta, Canada T6G 2P5. Electronic address: jwu3@ualberta.ca.
authors.forename: Jianping
authors.initials: J
authors.lastname: Wu
mesh_terms: D000818:Animals; D002645:Chickens / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015891:Cystatins / Q000302:isolation & purification*; D015853:Cysteine Proteinase Inhibitors / Q000302:isolation & purification*; D004529:Egg White / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D053719:Tandem Mass Spectrometry
chemical_list: D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; C100158:cystatin, egg-white
keywords: Affinity chromatography; Cystatin; HPLC; Hen egg white; Mass spectrometry; Quantification