Bacterial expression and characterization of chicken apolipoprotein A-I.

Apolipoprotein (apo) A-I is a 28-kDa exchangeable apolipoprotein that plays a key role in lipoprotein metabolism. It is widely distributed among animal species and is rich in alpha-helical secondary structure. Unlike human apoA-I, which aggregates in the absence of lipid, chicken apoA-I is monomeric in the lipid-free state. To take advantage of this physical characteristic, a bacterial expression system for production of recombinant chicken apoA-I has been developed. The cDNA-encoding chicken apoA-I was cloned into the pET expression vector under the regulation of the lac operon and transformed into Escherichia coli. Recombinant apoA-I protein recovered from the soluble fraction of the bacterial cell pellet was purified to greater than 95% homogeneity by reversed-phase high-performance liquid chromatography. Although immunoblot analysis confirmed the identity of the overexpressed protein, its migration on denaturing polyacrylamide gel electrophoresis was slower than its natural counterpart. To determine if the vector-encoded 18 residue pelB N-terminal leader sequence was not cleaved by the bacterial leader peptidase, isolated recombinant chicken apoA-I was incubated with exogenous leader peptidase. This treatment resulted in an increased electrophoretic mobility, with migration to a position corresponding to plasma-derived chicken apoA-I. Electrospray mass spectrometry indicated a mass of 27,961 +/- 4 Da, in agreement with that predicted for natural chicken apoA-I. Far-UV circular dichroism spectroscopy indicated an alpha-helical content similar to apoA-I isolated from chicken plasma, suggesting that the protein is folded in solution. Fluorescence studies showed that the wavelength of maximum fluorescence emission of the two tryptophan residues in the protein was 331 nm, with no shift occurring following complexation with lipid. Recombinant apoA-I was shown to be functional in lipoprotein binding as well as to possess an ability to transform bilayer vesicles of dimyristoylphosphatidylcholine into discoidal complexes. This is the first report of bacterial expression of an avian apoA-I. Increased availability and the potential for site-directed mutagenesis of this protein will aid in further characterization of apoA-I and the mechanism whereby it functions in cholesterol transport.