High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli

Abstract

Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lack of knowledge about its structure, specific biological function, and role in cardiovascular disease. Here we present a novel Escherichia coli-based recombinant expression system that produces highly pure mature human apoA-II at substantial yields. A Mxe GyrA intein containing a chitin binding domain was fused at the C terminus of apoA-II. A 6× histidine-tag was also added at the fusion protein's C terminus. After rapid purification on a chitin column, intein auto-cleavage was induced under reducing conditions, releasing a peptide with only one extra N-terminal Met compared with the sequence of human mature apoA-II. A pass through a nickel chelating column removed any histidine-tagged residual fusion protein, leaving highly pure apoA-II. A variety of electrophoretic, mass spectrometric, and spectrophotometric analyses demonstrated that the recombinant form is comparable in structure to human plasma apoA-II. Similarly, recombinant apoA-II is comparable to the plasma form in its ability to bind and reorganize lipid and promote cholesterol efflux from macrophages via the ATP binding cassette transporter A1. This system is ideal for producing large quantities of recombinant wild-type or mutant apoA-II for structural or functional studies.

Fig. 1.

pTWIN1 vector map showing the design of the apoA-II expression construct. Mature human apoA-II (blue) was subcloned into the pTWIN1 expression vector upstream of the T7 promoter with Mxe intein, a chitin binding domain, and a 6× histidine tag on its C terminus (green). To initiate transcription, a ATG codon was added to the N-terminus of the protein sequence. The site of intein-mediated endo-cleavage is indicated by a black arrow. The amino acid sequence of the translated apoA-II-intein fusion protein is reported: mature apoA-II (blue), modified Mxe intein (green).

Fig. 2.

Purification summary. A: SDS-glycine PAGE gel (4–20%) stained with Coomassie blue. Lane 1: Precision Plus protein standards from Bio-Rad with indicated molecular mass; lane 2: uninduced bacterial lysate; lane 3: IPTG-induced bacterial lysate. Amplified band of intein fusion protein is marked. B: SDS-tricine PAGE gel (10–20%) stained with Coomassie blue. Lane 1: Precision Plus protein standards from Bio-Rad; lane 2: chitin beads after activation of intein auto-cleavage reaction and column elution; lane 3: eluate from the chitin column after activation of intein auto-cleavage reaction; lanes 4 and 5: flow through fraction from a Ni-chelating affinity column in which residual fusion protein has been removed; reducing and nonreducing conditions, respectively. The final purity of recombinant apoA-II is >95% by combined SDS-PAGE and mass spectrometric analyses.

Fig. 3.

Top-down mass spectrometry analysis of human plasma purified and recombinant apoA-II. Protein samples were injected into a HPLC system equipped with a C18 column. The column flow-through was electrosprayed into a QStar XL mass spectrometer. Mass spectra at 18 min retention time are shown. Top: Human plasma apoA-II showing three major peaks. Bottom: Recombinant apoA-II showing a single major peak. Observed average masses resulting from the proteins are reported in Da. The mass accuracy of the instrumentation in this molecular mass range is ±2 Da.

Fig. 4.

Far UV circular dichroism spectra and Gnd-HCl denaturation. A: Far UV CD spectra of human plasma (solid) and recombinant (dashed) apoA-II. Samples were run at room temperature in triplicate for three accumulations each and reported as the overall mean. B: Isothermal Gnd-HCl denaturation of lipid-free human plasma (closed circles) and recombinant (open circles) apoA-II. Samples at 100 µg/ml were incubated at 4°C with the indicated concentration of Gnd-HCl for 72 h to ensure equilibrium. Average values and error bars from at least two separate experiments are reported.

Fig. 5.

Ability of human plasma and recombinant apoA-II to bind and reorganize DMPC liposomes. A and B show two separate experiments with independent preparations of protein (n = 1 each). All samples were run at 24.5°C, the transition temperature of DMPC, for 10 min with readings every 30 s. Closed triangles: DMPC liposomes only; closed circles: human plasma apoA-II; open circles: recombinant apoA-II. Panel C shows a 8–25% native polyacrylamide gel analysis of the end products of the reaction. Lane 1: Human plasma apoA-II; lane 2: recombinant apoA-II; lane 3: Amersham HMW molecular markers with hydrodynamic diameters expressed in Å. The gel was stained with Coomassie blue.

Fig. 6.

Ability of human plasma and recombinant apoA-II to promote cellular cholesterol efflux via ABCA1. The apoA-II samples were used as cholesterol acceptors from cholesterol-labeled RAW264.7 macrophages. The proteins were added to the media at concentrations in the 1–10 μg/ml range for 6 h. Percent efflux was calculated by dividing the efflux media counts by the total counts (n = 3; error bars represent 1 SD). Closed and open circles: Human plasma and recombinant apoA-II in the presence of cAMP, respectively. Closed and open triangles: Human plasma and recombinant apoA-II without cAMP, respectively.