Purification and HDL-like particle formation of apolipoprotein A-I after co-expression with the EDDIE mutant of Npro autoprotease

On the Aggregation of Apolipoprotein A-I

“…The expression and purification of ApoA-I was carried out as reported [ 41 ]. In summary, human ApoA-I was expressed in E. coli in fusion with the autoprotease N pro mutant EDDIE, on which a His-tag was added.…”

“…The inclusion bodies were dissolved in 4 M urea and the His-EDDIE refolded and cleaved from the target protein by dilution into 5 mM dithiothreitol (DTT), 10 mM Tris/HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0 and left on stirring for 48 h. The cleaved ApoA-I was purified through anionic ion exchange, immobilized metal affinity chromatography, hydrophobic interaction chromatography and boiling prior to being aliquoted, lyophilized and stored at −20 °C. Because the sample elutes in water at the last step, the lyophilized protein is salt-free except for the counter-ions [ 41 ]. The same protocol was used for wildtype ApoA-I, and a natural single point mutant, ApoA-I K107Δ [ 41 ].…”

“…Because the sample elutes in water at the last step, the lyophilized protein is salt-free except for the counter-ions [ 41 ]. The same protocol was used for wildtype ApoA-I, and a natural single point mutant, ApoA-I K107Δ [ 41 ].…”

“…Aliquots of purified ApoA-I were dissolved in 6 M GuHCl, and the monomer was isolated by size exclusion chromatography (SEC) on a 10 mm × 300 mm ENrich™ SEC70 column (Bio-Rad) in 20 mM sodium phosphate, 0.2 mM EDTA, pH 7.4 or 6.0. After the SEC step, no other proteins were observed by SDS PAGE [ 41 ]. The protein concentration was determined from the absorbance of either the integrated peak area or of the collected fraction using a NanoDrop 2000 instrument (Thermo Scientific) and the concentration determined from the baseline-corrected absorbance at 280 nm using ε 280 = 1.13 L g −1 cm −1 [ 42 ] and molecular weight 28 kDa.…”

Retardation of Aβ42 fibril formation by apolipoprotein A-I and recombinant HDL particles

“…The expression and purification of ApoA-I was carried out as previously reported ( 51 ). In summary, human ApoA-I was expressed in E. coli in fusion with the N pro autoprotease mutant EDDIE, onto which a His-tag was added.…”