{"id":{"repo_id":"ubc","oai_identifier":"oai:circle.library.ubc.ca:2429/2053"},"canonical_url":"https://search.dev.ndltd.org/etd/ubc/oai:circle.library.ubc.ca:2429/2053","repository":{"repo_id":"ubc","name":"University of British Columbia","base_url":"http://circle.library.ubc.ca/oai/request"},"display":{"title":"Use of the cellulose-binding domain of a cellulase from cellulomonas fimi for affinity purification of fusion preteins","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Greenwood, Jeffrey M."],"institution":"University of British Columbia","degree_name":"Doctor of Philosophy - PhD","degree_level":"doctoral","degree_discipline":"Microbiology and Immunology","degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":1993,"date_issued":"1993","date_published":"1993","updated_at":"2026-07-24T05:07:30Z","subjects":[],"languages":["eng"],"rights":["For non-commercial purposes only, such as research, private study and education. 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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."]},{"key":"dc:format","label":"Dc Format","values":["9884889","application/pdf"]},{"key":"dc:title","label":"Title","values":["Use of the cellulose-binding domain of a cellulase from cellulomonas fimi for affinity purification of fusion preteins"]}]}],"canonical_facts":{"dc:creator":["Greenwood, Jeffrey M."],"dc:date":["1993"],"dc: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. 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