2026-09-22T22:58:08Z https://www.ideals.illinois.edu/oai-pmh

oai:www.ideals.illinois.edu:2142/20792 2023-07-10 col_2142_5131 col_2142_14795 com_2142_5130 com_2142_14794 com_2142_14793 com_2142_8903

Jonas, Ana McGuire, Kirsten Arnvig 2011-05-07T12:49:25Z 2011-05-07T12:49:25Z 10000-01-01 1996 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. Made available in DSpace on 2011-05-07T12:49:25Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9702606.pdf: 7434770 bytes, checksum: a1fb074eee05edae5d9654626cd12da5 (MD5) Previous issue date: 1996 Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:46:18Z Item is restricted indefinitely. Restriction data tranferred 2014-07-01T11:20:42-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission ETDs are only available to UIUC Users without author permission U of I Only 9780591088762 AAI9702606 (UMI)AAI9702606 http://hdl.handle.net/2142/20792 eng Copyright 1996 McGuire, Kirsten Arnvig Biology, Animal Physiology Chemistry, Biochemistry Overexpression, purification and characterization of human proapolipoprotein A-I and mutants text Biochemistry Biochemistry University of Illinois at Urbana-Champaign Dissertation Ph.D.