Refolding and purification of a urokinase plasminogen activator fragment by chromatography

被引:38
作者
Fahey, EM [1 ]
Chaudhuri, JB
Binding, P
机构
[1] Univ Bath, Dept Chem Engn, Bath BA2 7AY, Avon, England
[2] Pfizer Ltd, Cent Res, Sandwich CT13 9NJ, Kent, England
来源
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES | 2000年 / 737卷 / 1-2期
关键词
Escherichia coli; refolding; purification; enzymes; urokinase plasminogen activator;
D O I
10.1016/S0378-4347(99)00360-6
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A fragment of recombinant urokinase plasminogen activator (u-PA), was expressed in E. coli in the form of inclusion bodies. Purification and renaturation was achieved in a three-stage process. Capture of the inclusion bodies was achieved by coupling wash steps in Triton X-100 and urea with centrifugation. Solubilised inclusion bodies were then renatured by buffer exchange performed by size-exclusion chromatography (SEPROS). Use of size-exclusion media with higher fractionation ranges resulted in an increase in the recovery of u-PA activity, to a maximum fractionation range of M-r 10 000-1 500 000 after which recovery is reduced, due to a low resolution between the refolded u-PA and denaturant. Fractions of refolded u-PA were concentrated using cation ion-exchange chromatography, which selectively binds correctly folded u-PA. The result is concentrated, active, homogeneous u-PA. (C) 2000 Elsevier Science B.V. All rights reserved.
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页码:225 / 235
页数:11
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