Bacterial overexpression, isotope enrichment, and NMR analysis of the N-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 expression vector and introduced into Escherichia coli. Induction of protein expression with isopropyl beta-D-thiogalactopyranoside resulted in production of recombinant apoE3(1-183). Immunoblot analysis revealed that recombinant protein was present in both the cell pellet and cell culture supernatant. Analysis revealed that a significant portion of the rApoE3(1-183) in the cell pellet still possessed the bacterial N-terminal pel B leader sequence, encoded by plasmid DNA directly upstream of the apoE3(1-183) coding sequence. By contrast, this hydrophobic leader sequence had been removed from recombinant protein specifically accumulating in the culture medium. This behavior is novel for bacterial expression of apolipoprotein E and its truncated variants and permits efficient overexpression of the recombinant protein (> 100 mg/L cell culture). Recombinant apoE3(1-183) was isolated by a combination of heparin-Sepharose chromatography and reverse-phase HPLC. Electrospray mass spectrometry provided a mass of 21 191 daltons, corresponding directly to that expected from the known sequence. Circular dichroism spectroscopy revealed that the recombinant protein possesses significant amounts of alpha-helical secondary structure. The lipid binding ability of rApoE3(1-183) was evaluated using an in vitro lipoprotein binding assay. It was observed that recombinant apoE3(1-183) protected human low density lipoprotein (LDL) from lipid accumulation induced particle aggregation, indicating that it is capable of associating with lipoprotein surfaces. In addition, rApoE3(1-183) forms disk complexes with the model phospholipid dimyristoylphosphatidylcholine. In competition experiments, it was observed that rApoE3(1-183) phospholipid disks compete with 125I-LDL for binding to the apoB/E receptor on human skin fibroblasts to an extent similar to that observed for intact rApoE3. Taken together, these data show that recombinant apoE3(1-183) is fully functional as an apolipoprotein and receptor ligand. Given the high expression level and its known existence as a monomer in solution, we evaluated the potential for application of NMR spectroscopy to study the structure-function relationship of rApoE3(1-183). Bacteria were cultured in media supplemented with 15NH4Cl or [15N]glycine and the isotopically labeled recombinant apoE3(1-183) was analyzed by heteronuclear single 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 association.
Fisher
C A
CA
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
Wang
J
J
Francis
G A
GA
Sykes
B D
BD
Kay
C M
CM
Ryan
R O
RO
D053318:Apolipoprotein E3; D001057:Apolipoproteins E / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D002942:Circular Dichroism; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012996:Solutions
D053318:Apolipoprotein E3; D001057:Apolipoproteins E; D011994:Recombinant Proteins; D012996:Solutions