Recombinant locust apolipophorin III: characterization and NMR spectroscopy

Abstract

Apolipophorin III (apoLp-III) from the locust Locusta migratoria is an exchangeable apolipoprotein that reversibly binds to lipoproteins. During lipid binding the protein has been proposed to undergo a major conformational change. To study the mechanism of lipid binding we have cloned and expressed recombinant protein in bacteria, permitting stable isotope enrichment for heteronuclear NMR spectroscopy and site-directed mutagenesis. The cDNA coding for apoLp-III was subcloned into the pET expression vector and transformed into Escherichia coli cells. Induction of expression resulted in the specific appearance of apoLp-III in the cell culture medium, indicating it escaped the bacteria without lysis. The protein was purified from the cell-free supernatant by reversed-phase HPLC, characterized and compared to the natural protein isolated from locust hemolymph. SDS-PAGE revealed the recombinant protein has a molecular mass of approximately 17 kDa, similar to that of deglycosylated natural apoLp-III. Monoclonal antibodies were used to detect recombinant apoLp-III in the cells as well as in cell-free medium of induced bacterial cultures. Amino acid sequencing and analysis confirmed the identity of the recombinant protein as L. migratoria apoLp-III. Circular dichroism spectroscopy of recombinant and natural apoLp-III showed similar spectra, both displaying high contents of α-helical secondary structure. Denaturation studies of lipid-free apoLp-III with guanidine hydrochloride showed that both proteins have similar denaturation midpoints and ΔG values indicating similar protein stability. The natural and recombinant protein were functional in lipoprotein binding assays. Using recombinant protein, uniformly and specifically labeled with ¹⁵N-amino acids, two dimensional ¹H-¹⁵N heteronuclear single quantum correlation spectra were obtained. The spectra revealed excellent chemical shift dispersion in both the ¹H and ¹⁵N dimensions with a well defined resonance pattern. Studies with ¹⁵N-leucine specifically labeled apoLp-III in the presence and absence of the micelle forming lipid, dodecylphosphocholine, provided evidence for a significant conformational change upon lipid association.