Heterogeneity determination and purification of commercial hen egg-white lysozyme

被引:125
|
作者
Thomas, BR
Vekilov, PG
Rosenberger, F
机构
[1] Center for Microgravity and Materials Research, University of Alabama in Huntsville, Huntsville
关键词
D O I
10.1107/S090744499600279X
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Hen egg-white lysozyme (HEWL) is widely used as a model protein, although its purity has not been adequately characterized by modern biochemical techniques. We have identified and quantified the protein heterogeneities in three commercial HEWL preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis with enhanced silver staining, reversed-phase fast protein liquid chromatography (FPLC) and immunoblotting with comparison to authentic protein standards. Depending on the source, the contaminating proteins totalled 1-6% (w/w) and consisted of ovotransferrin, ovalbumin, HEWL dimers, and polypeptides with approximate M(r) of 39 and 18 kDa. Furthermore, we have obtained gram quantities of electrophoretically homogeneous [> 99.9% (w/w)] HEWL by single-step semi-preparative scale cation-exchange FPLC with a yield of about 50%. Parallel studies of crystal growth kinetics, salt repartitioning and crystal perfection with this highly purified material showed fourfold increases in the growth-step velocities and significant enhancement in the structural homogeneity of HEWL crystals.
引用
收藏
页码:776 / 784
页数:9
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