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 $\alpha$-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.U of I OnlyETDs are only available to UIUC Users without author permissio