IMPROVED IMMOBILIZATION OF FUSION PROTEINS VIA CELLULOSE-BINDING DOMAINS

Cellulose-binding domains (CBDs) are structurally and functionally ind ependent, noncatalytic modules found in many cellulose or hemicellulos e degrading enzymes. Recent biotechnological applications of the CBDs include facilitated protein immobilization on cellulose supports. In s ome occasions there have been concerns about the stability of the CBD driven immobilization. Here we have studied the chromatographic behavi or of variants of the Trichoderma reesei cellobiohydrolase CBD belongi ng to family 1. Both CBDs fused to antibody fragments and isolated CBD s 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 leaka ge 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% pe r column volume. The improved immobilization could also be seen as a d ecreased migration of the protein down the column in extended washes. (C) 1998 John Wiley & Sons, Inc.