Monitoring the production of inclusion bodies during fermentation and enzyme-linked immunosorbent assay analysis of intact inclusion bodies using cryogel minicolumn plates

Research output: Contribution to journalArticle

Abstract

A novel minicolumn chromatgraphic method to monitor the production of inclusion bodies during fermentation and anenzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced its inclusion bodies wits labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous Monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed LIS to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed oil the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary I-G and of enzymatic reaction within the adsorbent. (c) 2006 Elsevier Inc. All rights reserved.

Details

Authors
  • Josefin Ahlqvist
  • Maria Dainiak
  • Ashok Kumar
  • E. Gunnar Hornsten
  • Igor Galaev
  • Bo Mattiasson
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Industrial Biotechnology

Keywords

  • minicolumn plate, monitoring, antibodies, protein A- and phenyl-cryogel, cryogel adsorbents, bioprocess, fermentation, inclusion bodies, ELISA, surface-expressed antigen
Original languageEnglish
Pages (from-to)229-237
JournalAnalytical Biochemistry
Volume354
Issue number2
Publication statusPublished - 2006
Publication categoryResearch
Peer-reviewedYes