Expression of N-acetyl-[beta]-D-Glucosaminidase of Bacteriodes forsythus
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Abstract
Bacteroides forsythus has been implicated in human periodontal diseases. This asachcharolytic bacteria expresses a number of glycolytic enzymes capable of hydrolyzing host proteoglycan and extracellular matrix proteins. In order to explore the role of these enzymes in periodontal tissue damage, a gene encoding for N-acetyl-f3-D glucosaminidase cloned from B. forsythus ATCC 43037, was cloned in to a plasmid expression vector, pET 22b (+) to over express the gene in E. coli and purify the enzyme.
The cloned N-acetyl-f3-D glucosaminidase gene was amplified with appropriate pnmers that facilitated its ligation in to the pET vector in frame and the ligated plasmid construct was transformed into E. coli MRF. The recombinant plasmid was transformed into an expression host E. coli BL21 (DE3). An enzymatic assay, SDS-PAGE and Western blot analysis confirmed that N acetyl-f3-D glucosaminidase gene has been cloned into pET vector and was under the induction of IPTG. Although the protein was over expressed in E. coli BL21 (DE3), purification of the enzyme was not successful. Also, levels of the protein increased after sonication implying that the protein was either in the cytoplasm or is bound to the membrane fraction of the cells. The difficulty associated with the purification of this protein indicates that this protein may be localized in the membrane fraction of the cells. Further studies might include methods to solubilize this fraction in order to facilitate purification. Future studies should examine other methods to increase the induction of this gene and facilitate its purification.
Description
Thesis (M.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 2001 (Pediatric Dentistry).
Includes bibliographical references (leaves 49-61).
Includes bibliographical references (leaves 49-61).
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This work is being made available in OpenBU by permission of its author, and is available for research purposes only. All rights are reserved to the author.