Expression and purification of non-prolyl cis-peptide bond mutants in human plasma platelet activating factor acetylhydrolase
University of Delaware
Platelet activating factor acetylhydrolase (PAF-AH) is a protein that is found in blood plasma and is associated with lipoproteins. PAF-AH is a calcium-independent serine esterase that functions on the lipid-aqueous interface and is known to degrade platelet-activating factor (PAF) by hydrolyzing the ester bond at the sn-2 position to produce the biologically inactive products lyso-PAF and acetate. In addition to PAF, PAF-AH is also capable of hydrolyzing other substrates, such as oxidized phosphatidylcholine. The cleavage of oxidized phosphatidylcholine produces lysophosphatidylcholine and oxidized nonesterified fatty acids, which are proinflammatory and proapoptotic lipid mediators. The buildup of these proinflammatory lipids is believed to support atherosclerotic plaque expansion, as well as the establishment and advancement of the necrotic centers of atherosclerosis. PAF-AH has a non-prolyl cis peptide bond between Phe-72 and Asp-73 that is located in a ?-hairpin type IV that extends from Ser-64 to Ser-87. The existence a non-prolyl cis peptide bond at this location serves no known purpose, however trans-cis isomerization may complicate the folding kinetics and create a transient population of partially folded intermediates. This heterogeneity in the unfolded state of the protein may give rise to a series of slow-folding reactions that dramatically slow the overall rate of protein folding and contribute to the high activation barrier of isomerization. In addition, protein heterogeneity may hinder the production of diffraction quality crystals. In this study, a series of three mutations were produced at the site of the non-prolyl cis peptide bond in an effort to create a more homogenous plasma PAF-AH protein sample and possibly increase the overall yield of pure protein. Furthermore, an additional series of mutations were produced to enhance the somanase activity of plasma PAF-AH. These mutations were made to facilitate the formation of a catalytic triad to aid in the hydrolysis of soman by plasma PAF-AH.