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Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies

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Author

  • Josefin Ahlqvist
  • Ashok Kumar
  • H Sundstrom
  • E Ledung
  • EG Hornsten
  • SO Enfors
  • B Mattiasson

Summary, in English

A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B.. 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.

Department/s

Publishing year

2006

Language

English

Pages

216-225

Publication/Series

Journal of Biotechnology

Volume

122

Issue

2

Document type

Journal article

Publisher

Elsevier

Topic

  • Industrial Biotechnology

Keywords

  • bodies
  • inclusion
  • cryogels
  • sulfamethazine
  • protein A
  • IgY
  • antibodies
  • IgG

Status

Published

ISBN/ISSN/Other

  • ISSN: 1873-4863