Overexpression, purification and characterization of human proapolipoprotein A-I and mutants

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 α-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 α-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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• McGuire, Kirsten Arnvig

Contributors dc:contributor

• Jonas, Ana

• Biology, Animal Physiology
• Chemistry, Biochemistry

Statement dc:rights

• Copyright 1996 McGuire, Kirsten Arnvig

Language dc:language

eng

Identifier

9780591088762
AAI9702606
(UMI)AAI9702606

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/20792