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Title: Conformation and activity of plasminogen activator inhibitor type 1 (PAI-1)
Author: Sancho, Elena
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1994
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Plasminogen activator inhibitor type 1 (PAI-1) is a unique member of the serpins in that it can adopt various conformations. It is synthesized as an active inhibitor which rapidly converts to a latent form that is not inhibitory. Conformational studies on this protein have been made difficult by this property. The production of recombinant PAI-1 was studied. The expression of r-PAI-1 by the bacterial strain MSD 1005 accounted for some 15-20% of the total cell protein, although some was produced in the form of inclusion bodies. As an attempt to improve the yields of PAI-1 antigen and activity, the cells were grown at low temperatures or under osmotic-stress conditions in the presence of betaine. These did not affect the distribution of r-PAI-1 between the soluble phase and inclusion bodies or the specific activity of the r-PAI-1. A quick protocol for the purification of r-PAI-1 from the strain MSD 1005 was developed. Approximately 26 mg of r-PAI-1 could be purified from 1.51 of bacterial culture in one step by ion exchange chromatography. The specific activity of the purified r-PAI-1 was found to be close to the theoretical maximum and to be unusually stable. The stability of the r-PAI-1 was found to be dependent on the low pH and the high NACl concentration used during the purification protocol. The effect of arginine was found to be comparable to that of NaCl. Vitronectin or aprotinin had no effect on the specific activity of r-PAI-1. Active, denaturant activated and latent r-PAI-1 were compared in terms of their binding to heparin and to a panel of monoclonal antibodies. Complex formation with t-PA was also assessed. Active and denaturant activated PAI-1 were indistinguishable in these studies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry Biochemistry Molecular biology Cytology Genetics