PURIFICATION AND PROCESSING OF CELLULOSE-BINDING DOMAIN-ALKALINE PHOSPHATASE FUSION PROTEINS

Fusion of the leader peptide and the cellulose-binding domain (CBD) of endoglucanase A (CenA) from Cellulomonas fimi, with or without linker sequences, to the N-terminus of alkaline phosphatase (PhoA) from Esch erichia coil leads to the accumulation of significant amounts of the C BD-PhoA fusion proteins in the supernatants of E. coli cultures. The f usion proteins can be purified from the supernatants by affinity chrom atography on cellulose. The fusion proteins can be desorbed from the c ellulose with water or guanidine-HCl. If the sequence IEGR is present between the CBD and PhoA, the CBD can be cleaved from the PhoA with fa ctor Xa. The efficiency of hydrolysis by factor Xa is strongly influen ced by the amino acids on either side of the IEGR sequence. The CBD re leased by factor Xa is removed by adsorption to cellulose. A nonspecif ic protease from C. fimi, which hydrolyzes native CenA between the CBD and the catalytic domain, may be useful for removing the CBD from som e fusion proteins. (C) 1994 John Wiley & Sons, Inc.