Henrik Ferré

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

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 β2‐microglobulin and a binding peptide; and (2) optimal in vitro conditions for disulfide bond formation (∼pH 8) and peptide binding (∼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 β2‐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.

A novel system for continuous protein refolding and on‐line capture by expanded bed adsorption

A novel two‐step protein refolding strategy has been developed, where continuous renaturation‐bydilution is followed by direct capture on an expanded bed adsorption (EBA) column. The performance of the overall process was tested on a N‐terminally tagged version of human β2‐microglobulin (HAT‐hβ2m) both at analytical, small, and preparative scale. In a single scalable operation, extracted and denatured inclusion body proteins from Escherichia coli were continuously diluted into refolding buffer, using a short pipe reactor, allowing for a defined retention and refolding time, and then fed directly to an EBA column, where the protein was captured, washed, and finally eluted as soluble folded protein. Not only was the eluted protein in a correctly folded state, the purity of the HAThβ2m was increased from 34% to 94%, and the product was concentrated sevenfold. The yield of the overall process was 45%, and the product loss was primarily a consequence of the refolding reaction rather than the EBA step. Full biological activity of HAT‐hβ2m was demonstrated after removal of the HAT‐tag. In contrast to batch refolding, a continuous refolding strategy allows the conditions to be controlled and maintained throughout the process, irrespective of the batch size; i.e., it is readily scalable. Furthermore, the procedure is fast and tolerant toward aggregate formation, a common complication of in vitro protein refolding. In conclusion, this system represents a novel approach to small and preparative scale protein refolding, which should be applicable to many other proteins.