Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.656385
Title: Characterisation and purification of an aggrecanase made by injured synovium
Author: Rana, Faisal
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Abstract:
Freshly dissected porcine synovial tissue in culture produces an enzymatic activity that cleaves cartilage aggrecan generating ARGS- and AGEG- bearing neo-epitope fragments. The aggrecanolytic activity was abolished when synovial tissue was cultured in the presence of cycloheximide. The enzyme(s) were sensitive to N-terminal inhibitory domain of tissue inhibitor of matrix metalloproteinase (N-TIMP-3) and general matrix metalloproteinase inhibitor (GM6001) suggesting they may belong to a disintegrin and metalloproteinase (ADAM) or ADAM with thrombospondin motifs (ADAMTS) family of enzymes. Cation exchange chromatography was used to partially purify aggrecanase(s) from synovial tissue culture medium (SYCM). Two active species have been separated from the partially purified material using size-exclusion chromatography. The smaller species had a molecular weight of 35-40 kDa while the larger enzyme had an apparent molecular weight greater than 2000 kDa. Low density lipoprotein receptor-related protein (LRP1) didn't appear to be involved in the formation of higher molecular weight complex. The smaller species was further chromatographed on a SMART mono Q column. The sequential chromatography gave approximately 400-fold enrichment of the enzyme. The concentration of the enzyme was estimated by titration with recombinant N-TIMP-3, which was expressed and purified from E.coli. The N-TIMP-3 was electrostatically coupled to Ni2+ agarose beads. The beads were then used to affinity purify the enzyme from mono Q fractions. The affinity-purified material was electrophoresed and protein bands were selected for mass spectrometry. No ADAMTS enzyme was identified in the candidate bands. Further improvements will be made to the purification procedure to identify the synovial aggrecanase.
Supervisor: Saklatvala, Jeremy; Nagase, Hideaki Sponsor: Kennedy Trust for Rheumatology Research
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.656385  DOI: Not available
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