Many different assays for measuring peptide-MHC interactions have been suggested over the years. Yet, there is no generally accepted standard method available. We have recently generated preoxidized recombinant MHC class I molecules (MHC-I) which can be purified to homogeneity under denaturing conditions (i.e., in the absence of any contaminating peptides). Such denatured MHC-I molecules are functional equivalents of "empty molecules". When diluted into aqueous buffer containing beta-2 microglobulin (beta2m) and the appropriate peptide, they fold rapidly and efficiently in an entirely peptide dependent manner. Here, we exploit the availability of these molecules to generate a quantitative ELISA-based assay capable of measuring the affinity of the interaction between peptide and MHC-I. This assay is simple and sensitive, and one can easily envisage that the necessary reagents, standards and protocols could be made generally available to the scientific community.

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 ␤ 2 -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 ␤ 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.Keywords: MHC class I; protein folding; disulfide bond formation; inclusion bodies; hydrophobic interaction chromatography Major histocompatibility complex class I (MHC-I) molecules are expressed on the surface of almost all cells in the human body. These molecules are ternary complexes consisting of three components: (1) a glycosylated heavy chain (44 kD), containing two disulfide bonds; (2) a noncovalently associated light chain, ␤ 2 -microglobulin (12 kD), containing a single disulfide bond; and (3) a tightly bound peptide (Springer et al. 1977). Their function is to sample endogenously derived peptides, transport them to the cell surface, and present them to cytotoxic T cells, which continuously scan cell surfaces for peptide-MHC-I complexes. Peptides presented in context with MHC-I molecules originate from the digestion of intracellular proteins, normal ones and those of pathogens. MHC-I molecules therefore serve as a link between the intracellular compartment, which is inaccessible to the cells of the immune system, and the extracellular compartment, in which the immune cells Abbreviations: ␤ 2 m, ␤ 2 -microglobulin; CV, column volumes; HIC, hydrophobic interaction chromatography; HLA, human leucocyte antigen; IB, inclusion bodies; MHC-I, major histocompability complex class I; SEC, size exclusion chromatography.Article and publication are at http://www.proteinscience.org/cgi

An arabinoxylan arabinofuranohydrolase was isolated from barley malt. The enzyme preparation, Ara 1, contained two polypeptides with apparent molecular masses of < 60 and < 66 kDa, a pI of 4.55 and almost identical N-terminal amino-acid sequences. With p-nitrophenyl a-l-arabinofuranoside (pNPA) as substrate, Ara 1 exhibited a K m of 0.5 mm and a V max of 6.7 mmol´min 21´( mg of protein) 21 . Maximum activity was displayed at pH 4.2 and 60 8C, and, under these conditions, the half-life of the enzyme was 8 min. The Ara 1 preparation showed no activity against p-nitrophenyl a-l-arabinopyranoside or p-nitrophenyl b-d-xylopyranoside. Substrate preference and specificity were investigated using pure oligosaccharides and analysis by TLC and nano-probe NMR. Ara 1 released arabinose from high-molecular-mass arabinoxylan and arabinoxylan-derived oligosaccharides but was inactive against linear or branched-chain arabinan. Arabinose was readily released from both singly and doubly substituted xylo-oligosaccharides. Whereas single 2-O-linked and 3-O-linked arabinose substituents on non-reducing terminal xylose were released at similar rates, there was a clear preference for 2-Olinked arabinose on internal xylose residues. When Ara 1 acted on oligosaccharides with doubly substituted, non-reducing terminal xylose, the 3-O-linked arabinose group was preferred as the initial point of attack. Oligosaccharides with doubly substituted internal xylose were poor substrates and no preference could be determined. The enzyme described here is the first reported arabinoxylan arabinofuranohydrolase which is able to release arabinose from both singly and doubly substituted xylose, and it hydrolyses p-nitrophenyl a-l-arabinofuranoside at a rate similar to that observed for oligosaccharide substrates.

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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 b 2 -microglobulin (HAT-hb 2 m) 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 HAThb 2 m 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-hb 2 m 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.