Separation of fibronectin from a plasma gelatinase using immobilized metal affinity chromatography

FEBS Letters

Sparrman, Marianne¹  Smilenov, Lubomir⁴  Johansson, Staffan²  Zeligman, Ilia³  Forsberg, Erik² 

[1] Pharmacia Bio Process Technology AB, S-751 82 Uppsala, Sweden;Department of Medical and Physiological Chemistry, University of Uppsala, Biomedical Center, Box 575, S-751 23 Uppsala, Sweden;Pharmacia-LKB Biotechnology, BD 1-3 Uppsala, Sweden;Bulgarian Academy of Sciences, Central Laboratory of Biophysics, Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria

关键词: Fibronectin;    Gelatinase;    Immobilised metal affinity chromatography;    Zymography;    Chromatofocussing;    IMAC;    immobilized metal affinity chromatography;    PBE;    polybuffer exchanger;    PAGE;    polyacrylamide gel electrophoresis;    SDS;    sodium dodecyl sulfate;   

DOI  :  10.1016/0014-5793(92)80447-O

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Conventional preparations of plasma fibronectin are known to contain a co-purifying gelatinase [1986, J. Biol. Chem. 261, 4363–4366], but so far useful methods to remove the protease have not been available. In this study a number of different methods were tested in order to achieve separation of the two proteins. Immobilized metal affinity chromatography was found to be efficient for this purpose, and a convenient procedure to separate the two proteins under nondenaturing conditions on chelating Sepharose charged with Co²⁺, Ni²⁺, or Zn²⁺ is described. An alternative method employing pH gradient elution of an Fe³⁺ gel also resolved fibronectin from the gelatinase. The Fe³⁺ gel bound both proteins at pH 6.0 but not at pH 7.4, suggesting that the two proteins were phosphorylated. The described procedures will now allow studies of the functions of fibronectin in the absence of the contaminating protease.

【 授权许可】

Unknown