BACTERIAL OVEREXPRESSION, ISOTOPE ENRICHMENT, AND NMR ANALYSIS OF THEN-TERMINAL DOMAIN OF HUMAN APOLIPOPROTEIN-E

The nucleotide sequence encoding the N-terminal domain (residues 1-183 ) of human apolipoprotein E3 (apoE3) was cloned into the pET expressio n vector and introduced into Escherichia coli. Induction of protein ex pression with isopropyl beta-D-thiogalactopyranoside resulted in produ ction of recombinant apoE3(1-183). Immunoblot analysis revealed that r ecombinant protein was present in both the cell pellet and cell cultur e supernatant. Analysis revealed that a significant portion of the rAp oE3(1-183) in the cell pellet still possessed the bacterial N-terminal pel B leader sequence, encoded by plasmid DNA directly upstream of th e apoE3(1-183) coding sequence. By contrast, this hydrophobic leader s equence had been removed from recombinant protein specifically accumul ating in the culture medium. This behavior is novel for bacterial expr ession of apolipoprotein E and its truncated variants and permits effi cient overexpression of the recombinant protein (>100 mg/L cell cultur e). Recombinant apoE3(1-183) was isolated by a combination of heparin- Sepharose chromatography and reverse-phase HPLC. Electrospray mass spe ctrometry provided a mass of 21 191 daltons, corresponding directly to that expected from the known sequence. Circular dichroism spectroscop y revealed that the recombinant protein possesses significant amounts of alpha-helical secondary structure. The lipid binding ability of rAp oE3(1-183) was evaluated using an in vitro lipoprotein binding assay. It was observed that recombinant apoE3(1-183) protected human low dens ity lipoprotein (LDL) from lipid accumulation induced particle aggrega tion, indicating that it is capable of associating with lipoprotein su rfaces. In addition, rApoE3(1-183) forms disk complexes with the model phospholipid dimyristoylphosphatidylcholine. In competition experimen ts, it was observed that rApoE3(1-183) phospholipid disks compete with I-125-LDL for binding to the apoB/E receptor on human skin fibroblast s to an extent similar to that observed for intact rApoE3. Taken toget her, these data show that recombinant apoE3(1-183) is fully functional as an apolipoprotein and receptor ligand. Given the high expression l evel and its known existence as a monomer in solution, we evaluated th e potential for application of NMR spectroscopy to study the structure -function relationship of rApoE3(1-183). Bacteria were cultured in med ia supplemented with (NH4Cl)-N-15 or [N-15]glycine and the isotopicall y labeled recombinant apoE3(1-183) was analyzed by heteronuclear singl e quantum correlation NMR spectroscopy. The data revealed that rApoE3( 1-183) is an excellent candidate for solution structure studies by NMR , including conformational adaptations associated with lipid associati on.