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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.
Item Metadata
Title |
Use of the cellulose-binding domain of a cellulase from cellulomonas fimi for affinity purification of fusion preteins
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1993
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Description |
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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Extent |
9884889 bytes
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Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2008-09-16
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0098859
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1993-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.