Improved immobilization of fusion proteins via cellulose-binding domains

Research output: Contribution to journalArticle

Researchers

  • Markus Linder

  • Tarja K. Nevanen
  • Lotta Söderholm
  • Outi Bengs
  • Tuula T. Teeri

Research units

  • VTT Technical Research Centre of Finland

Abstract

Cellulose-binding domains (CBDs) are structurally and functionally independent, noncatalytic modules found in many cellulose or hemicellulose degrading enzymes. Recent biotechnological applications of the CBDs include facilitated protein immobilization on cellulose supports. In some occasions there have been concerns about the stability of the CBD driven immobilization. Here we have studied the chromatographic behavior of variants of the Trichoderma reesei cellobiohydrolase CBD belonging to family 1. Both CBDs fused to antibody fragments and isolated CBDs were studied and compared. Tritium labeling by reductive methylation was used as a sensitive detection method. The fusion protein as well as the isolated CBD was found to leak from the column at a rate of 0.3-0.5% of the immobilized protein per column volume. However, the leakage could be overcome by using two CBDs instead of a single CBD for the immobilization. In this way leakage was reduced to less than 0.01% per column volume. The improved immobilization could also be seen as a decreased migration of the protein down the column in extended washes. (C) 1998 John Wiley & Sons, Inc.

Details

Original languageEnglish
Pages (from-to)642-647
Number of pages6
JournalBiotechnology and Bioengineering
Volume60
Issue number5
Publication statusPublished - 5 Dec 1998
MoE publication typeA1 Journal article-refereed

    Research areas

  • cellulose, protein immobilization, affinity chromatography, cellulose-binding domain, Trichoderma reesei, NUCLEAR-MAGNETIC-RESONANCE, CRYSTALLINE CELLULOSE, TRICHODERMA-REESEI, CELLOBIOHYDROLASE-I, ADSORPTION, CELLULASES, REVERSIBILITY, EXCHANGE, FUNGAL, FIMI

ID: 2068313