Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding |
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| Authors: | Ferré Henrik Ruffet Emmanuel Blicher Thomas Sylvester-Hvid Christina Nielsen Lise Lotte B Hobley Timothy J Thomas Owen R T Buus Søren |
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| Affiliation: | Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, DK-2200, Copenhagen N, Denmark. |
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| 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. |
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| Keywords: | MHC class I protein folding disulfide bond formation inclusion bodies hydrophobic interaction chromatography |
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