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Use of the cellulose-binding domain of a cellulase from cellulomonas fimi for affinity purification of fusion preteins Greenwood, Jeffrey M.

Abstract

This study describes the use of a cellulose-binding domain (CBD) from a bacterial cellulase as an affinity tag for purification of heterologous proteins. The CBD of endoglucanase A (CenA) from Cellulomonas fimi is an N-terminal domain comprising amino acids. CenA binds strongly to cellulose, and the CBD retains this function when separated from its cognate catalytic domain by proteolysis or genetic manipulation. A series of fusions between CenA and alkaline phosphatase from Escherichia coli (PhoA) were generated using TnphoA, and were screened for binding to cellulose. CenA-PhoA fusion proteins containing an intact CBD bind to cellulose, while those missing 28 or 68 C-terminal amino acids from the CBD do not. Similarly, deletions of 18 or 44 amino acids from the N-terminus of the CBD abolish binding to cellulose. This is the first demonstration of a CBD retaining its function when fused to a heterologous polypeptide. Just one CBD is sufficient to bind dimeric alkaline phosphatase to cellulose. Engineered CenA-PhoA fusion proteins are purified in a single step by affinity chromatography on cellulose, with distilled water elution. The CBD was removed by specific proteolytic cleavage with Factor Xa or by a C. fimi serine protease. CBD fusions with human interleukin 2 (IL2) were constructed, but are predominantly insoluble and extensively degraded on expression in E. coli. However, the fusion polypeptides can still be purified by binding to cellulose.

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