Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding

Abstract

The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1). correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2). optimal in vitro conditions for disulfide bond formation ( approximately pH 8) and peptide binding ( approximately pH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules with correct disulfide bonding are formed under non-reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond-assisted folding in the presence of beta(2)-microglobulin and a specific peptide. Under conditions optimized for peptide binding, refolding and simultaneous peptide binding of the correctly oxidized heavy chain was much more efficient than that of the fully reduced molecule.

Figure 1.

Reducing SDS-PAGE analysis of expression levels of MHC-I heavy chains in Escherichia coli. (A) Expression levels of rA2. (B) Expression levels of rK^k (des cys). (C) Expression levels of rA11. Fermentor samples (15 μL) were withdrawn before and every hour after induction. The bacterial cell pellet was resuspended in 50 μL MgCl₂/SDS lysis buffer to release and solubilize heavy-chain inclusion bodies as described by Chen and Christen (1997). After centrifugation at 20,000g for 2 min, 15 μL of the supernatant was loaded directly on the gel. Lane 1, protein marker; lane 2, before induction; and lanes 3–5, samples taken 1, 2, and 3 h after induction with IPTG, respectively. Positions of heavy-chain monomers are shown with arrows.

Figure 2.

Reducing and non-reducing SDS-PAGE analysis of solubilized inclusion body preparations. (A) Solubilized inclusion bodies containing rA2. (B) Solubilized inclusion bodies containing rA11. The intensity of the heavy-chain monomer band increased on reduction, owing to the release of disulfide bond cross-linked monomers, and made the distinction of the isomers difficult. To improve the visualization of heavy-chain isomers, reducing samples of rA2 and rA11 were diluted twice as much in sample buffer as the non-reducing counterparts. The positions of heavy-chain isomers 0, 1, and 2 are indicated on the figure. NR indicates non-reducing; R indicates reducing.

Figure 3.

Separation of rA2 heavy-chain isomers by hydrophobic interaction chromatography on phenyl Sepharose High Performance under non-reducing denaturing conditions. Aliquots (7.5 μL) of fractions collected in the shaded area of the chromatogram were analyzed by non-reducing SDS-PAGE, and the result is shown below. Analyzed fractions are indicated on the figure, as well as the positions of heavy-chain isomers. Lane M, protein marker; lane L, sample applied on the hydrophobic interaction chromatography column.

Figure 4.

Separation of rA11 heavy-chain isomers by hydrophobic interaction chromatography on phenyl Sepharose High Performance under non-reducing denaturing conditions. Aliquots (12 μL) of fractions collected in the shaded area of the chromatogram were analyzed by non-reducing SDS-PAGE, and the result is shown below. Analyzed fractions are indicated on the figure, as well as the positions of heavy-chain isomers. Lanes M, protein marker; lane L, sample applied on the hydrophobic interaction chromatography column.

Figure 5.

Refolding and peptide binding analysis of fractionated MHC-I heavy chain isomers. (A) Analysis of fractions collected during purification of rA2 on phenyl Sepharose High Performance (see Fig. 3 ▶). (B) Analysis of fractions collected during purification of rA11 on phenyl Sepharose High Performance (see Fig. 4 ▶). Folding was initiated by diluting aliquots (1 μL) from selected fractions 100-fold in 100 mM Tris-maleate (pH 6.6) buffer, containing human β₂m (3 μM) and a radiolabeled peptide (15,000 cpm). The pixels intensities of heavy-chain isomers 1 and 2 were calculated from a densitometric analysis of SDS–polyacrylamide gels shown in Figure 3 and Figure 4 ▶ ▶, respectively. Fraction numbers are shown on the figure. Empty squares indicate isomer 1 protein tracing; solid squares, isomer 2 protein tracing; and circles, mean peptide binding. The standard deviation of duplicate peptide binding measurements was typically within 5%.

Figure 6.

Determination of the amount of oxidized rA11 heavy-chain monomer that refolds properly into the matured state with a quantitative and conformationally sensitive ELISA. The double log plot shows the amount of heavy-chain monomer offered to the folding reaction that was detected in the fully matured MHC-I complex. A non-reducing SDS-PAGE analysis of the purified rA11 sample is shown in the insert. Graded concentrations of denatured rA11 were diluted in a 100 mM Tris-maleate (pH 6.6) refolding buffer containing an excess of human β₂m (3 μM) and a specific peptide (10,000 nM). Detection of properly folded complexes and conversion of measured O.D.₄₅₀ values to picomolar complex was performed as described in Materials and methods. Lane 1, rA11 sample; lane 2, protein marker. Positions of rA11 isomers 1 and 3 are shown with arrows. Standard deviations of triplicate measurements are indicated on the figure.

Figure 7.

Dose-response curve for purified rA11 isomer 0 (reduced) and isomer 1 (oxidized). Graded concentrations of purified A11 isomer 0 and 1 were diluted 100-fold into 100 mM Tris-maleate (pH 6.6) buffer containing human β₂m (3 μM) and a specific radiolabeled peptide (15,000 cpm) and 1 mg/mL pluriol. The mean peptide binding values were calculated as described in Materials and Methods. Empty squares indicate rA11 isomer 1; solid squares, rA11 isomer 0. The standard deviation of duplicate peptide binding measurements was typically within 5%.