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Recovery of urokinase from integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using p-aminobenzamidine affinity chromatography

Bansal, Vibha ; Roychoudhury, Pradip K. ; Mattiasson, Bo LU and Kumar, Ashok LU (2006) In Journal of Molecular Recognition 19(4). p.332-339
Abstract
An integrated product recovery system was developed to separate urokinase from the cell culture broth of human kidney cells HT1080. Supermacroporous monolithic cryogels provided ideal matrices with respect to surface and flow properties for use as cell culture scaffold as well as for affinity chromatographic capture step of the enzyme in the integrated system. The urokinase was produced continuously in the reactor running for 4 weeks with continuous circulation of 500 ml of culture medium. The enzyme activity in the culture medium reached to 280 Plough units (PU)/mg protein. Cu(II)-iminodiacetic acid (IDA)-polyacrylamide (pAAm) cryogel column was used to capture urokinase by integrating with the gelatin-coupled pAAm-cryogel bioreactor for... (More)
An integrated product recovery system was developed to separate urokinase from the cell culture broth of human kidney cells HT1080. Supermacroporous monolithic cryogels provided ideal matrices with respect to surface and flow properties for use as cell culture scaffold as well as for affinity chromatographic capture step of the enzyme in the integrated system. The urokinase was produced continuously in the reactor running for 4 weeks with continuous circulation of 500 ml of culture medium. The enzyme activity in the culture medium reached to 280 Plough units (PU)/mg protein. Cu(II)-iminodiacetic acid (IDA)-polyacrylamide (pAAm) cryogel column was used to capture urokinase by integrating with the gelatin-coupled pAAm-cryogel bioreactor for HT1080 cell culture. After removing the urokinase capture column from the integrated system the bound protein was eluted. The metal affinity capture step gave 4.5-fold purification of the enzyme thus achieving a specific activity of 1300 PU/mg protein. The enzyme eluate from Cu(11)-IDA-pAAm cryogel capture column was further purified on benzamidine-Sepharose affinity column. This step finally led to a homogeneous preparation of different forms of urokinase in two different ellution peaks with a best urokinase activity of 13 550 PU/mg of protein. As compared to initial activity in the cell culture broth, about 26.2- and 48.4-fold increase in specific activity was achieved with enzyme yields corresponding to 32% and 35% in two different peak fractions, respectively. Native electrophoresis and SDS-PAGE showed multiple protein bands corresponding to different forms of the urokinase, which were confirmed by Western blotting and zymography. Copyright (c) 2006 John Wiley & Sons, Ltd. (Less)
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author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
integrated product recovery, cryogel matrix, urokinase, supermacroporous cryogels, p-aminobenzamidine chromatography, IMAC
in
Journal of Molecular Recognition
volume
19
issue
4
pages
332 - 339
publisher
John Wiley and Sons Inc.
external identifiers
  • wos:000239882300012
  • scopus:33747254428
  • pmid:16761300
ISSN
1099-1352
DOI
10.1002/jmr.785
language
English
LU publication?
yes
id
43053712-16fd-4495-8aa2-6bf212fce260 (old id 397378)
date added to LUP
2016-04-01 12:29:26
date last changed
2020-09-23 02:55:52
@article{43053712-16fd-4495-8aa2-6bf212fce260,
  abstract     = {An integrated product recovery system was developed to separate urokinase from the cell culture broth of human kidney cells HT1080. Supermacroporous monolithic cryogels provided ideal matrices with respect to surface and flow properties for use as cell culture scaffold as well as for affinity chromatographic capture step of the enzyme in the integrated system. The urokinase was produced continuously in the reactor running for 4 weeks with continuous circulation of 500 ml of culture medium. The enzyme activity in the culture medium reached to 280 Plough units (PU)/mg protein. Cu(II)-iminodiacetic acid (IDA)-polyacrylamide (pAAm) cryogel column was used to capture urokinase by integrating with the gelatin-coupled pAAm-cryogel bioreactor for HT1080 cell culture. After removing the urokinase capture column from the integrated system the bound protein was eluted. The metal affinity capture step gave 4.5-fold purification of the enzyme thus achieving a specific activity of 1300 PU/mg protein. The enzyme eluate from Cu(11)-IDA-pAAm cryogel capture column was further purified on benzamidine-Sepharose affinity column. This step finally led to a homogeneous preparation of different forms of urokinase in two different ellution peaks with a best urokinase activity of 13 550 PU/mg of protein. As compared to initial activity in the cell culture broth, about 26.2- and 48.4-fold increase in specific activity was achieved with enzyme yields corresponding to 32% and 35% in two different peak fractions, respectively. Native electrophoresis and SDS-PAGE showed multiple protein bands corresponding to different forms of the urokinase, which were confirmed by Western blotting and zymography. Copyright (c) 2006 John Wiley & Sons, Ltd.},
  author       = {Bansal, Vibha and Roychoudhury, Pradip K. and Mattiasson, Bo and Kumar, Ashok},
  issn         = {1099-1352},
  language     = {eng},
  number       = {4},
  pages        = {332--339},
  publisher    = {John Wiley and Sons Inc.},
  series       = {Journal of Molecular Recognition},
  title        = {Recovery of urokinase from integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using p-aminobenzamidine affinity chromatography},
  url          = {http://dx.doi.org/10.1002/jmr.785},
  doi          = {10.1002/jmr.785},
  volume       = {19},
  year         = {2006},
}