Greenwood J M, Gilkes N R, Miller R C, Kilburn D G, Warren R A
Department of Microbiology and Immunology and Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, BC, Canada.
Biotechnol Bioeng. 1994 Dec;44(11):1295-305. doi: 10.1002/bit.260441105.
Fusion of the leader peptide and the cellulose-binding domain (CBD) of endoglucanase A (CenA) from Cellulomonas fimi, with of without linker sequences, to the N-terminus of alkaline phosphatase (PhoA) from Escherichia coli leads to the accumulation of significant amounts of the CBD-PhoA fusion proteins in the supernatants of E. coli cultures. The fusion proteins can be purified from the supernatants by affinity chromatography on cellulose. The fusion protein can be desorbed from the cellulose with water or guanidine-HCl. If the sequence IEGR in present between the CBD and PhoA, the CBD can be cleaved from the PhoA with factor Xa. The efficiency of hydrolysis by factor Xa is strongly in fluenced by the amino acids on either side of the IEGR sequence. The CBD released by factor Xa is removed by adsorption to cellulose. A nonspecific proteases from C. fimi, which hydrolyzes native CenA between the CBD and the catalytic domain, may be useful for removing the CBD from some fusion proteins.
将来自纤维单胞菌的内切葡聚糖酶A（CenA）的前导肽和纤维素结合结构域（CBD）（有无接头序列）与大肠杆菌碱性磷酸酶（PhoA）的N端融合，会导致在大肠杆菌培养物的上清液中积累大量的CBD-PhoA融合蛋白。融合蛋白可通过纤维素亲和层析从上清液中纯化。融合蛋白可用水或盐酸胍从纤维素上解吸下来。如果CBD和PhoA之间存在IEGR序列，则CBD可被Xa因子从PhoA上切割下来。Xa因子的水解效率受到IEGR序列两侧氨基酸的强烈影响。Xa因子释放的CBD通过吸附到纤维素上而被去除。来自纤维单胞菌的一种非特异性蛋白酶，可在CBD和催化结构域之间水解天然CenA，可能有助于从某些融合蛋白中去除CBD。
