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|Title:||Overexpression, purification and characterization of human proapolipoprotein A-I and mutants|
|Author(s):||McGuire, Kirsten Arnvig|
|Doctoral Committee Chair(s):||Jonas, Ana|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Subject(s):||Biology, Animal Physiology
|Abstract:||The cDNA coding the human proapoA-I was cloned into an Escherichia coli vector, overexpressed and purified to 99% homogeneity and characterized together with apoA-I purified from human plasma. SDS-PAGE, mass spectrometry and Edman sequence analysis showed that the initial Met residue is post translationally removed. The proapoA-I self associated, interacted with dimyristoyl phosphatidylcholine vesicles and formed secondary structures similar to the lipid-free apoA-I. Reconstituted HDL particles made with phospholipid and cholesterol by the Na-cholate method had identical particle sizes, distributions and contained the same number of apoproteins per particle when apoA-I or proapoA-I were used. Furthermore, their $\alpha$-helical contents were the same, they had similar fluorescence properties and activated LCAT equally well. In conclusion, proapoA-I expressed and purified from E. coli is functionally and structurally indistinguishable from apoA-I purified from plasma when analyzed in vitro. Several proapoA-I mutants were constructed, purified and characterized. The deletion mutant proapoA-I$\Delta$187-217, was purified and the molecular weight was determined by mass spectrometry to be 25462 Da. Cross-linking of the mutant showed that it primarily existed as a monomer, but could form dimers. The association with DMPC liposomes was significantly reduced, but the mutant protein was able to form rHDL particles by the Na-cholate method. These particles had smaller sizes, and a reduced $\alpha$-helix content, but were equally stable to GdnHCl denaturation when compared to rHDL containing wild-type proapoA-I. The reactivity with LCAT was reduced by 5-fold.
Two proapoA-I point mutants proapoA-ID9C and proapoA-IW-3:8:50:72F, were purified and the molecular weight determined, to 28866 Da and 28727 Da, respectively, in agreement with the calculated molecular weight without the initial Met residues residue. The proapoA-ID9C mutant protein was able to form rHDL particles by the Na-Cholate method, these particles were used to determine size and shape of the rHDL particles by atomic force microscopy.
|Rights Information:||Copyright 1996 McGuire, Kirsten Arnvig|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9702606|