A Soluble Major Histocompatibility Complex Class I Peptide-binding Platform Undergoes a Conformational Change in Response to Peptide Epitopes*

Elizabeth Rigney, Mayumi Kojima, Ann Glithero, and Tim ElliottDagger

From the Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Class I major histocompatibility complexes (MHC) are heterotrimeric structures comprising heavy chains (HC), beta 2-microglobulin (beta 2-m), and short antigenic peptides of 8-10 amino acids. These components assemble in the endoplasmic reticulum and are released to the cell surface only when a peptide of the appropriate length and sequence is incorporated into the structure. The binding of beta 2-m and peptide to HC is cooperative, and there is indirect evidence that the formation of a stable heterotrimer from an unstable HC:beta 2-m heterodimer involves a peptide-induced conformational change in the HC. Such a conformational change could ensure both a strong interaction between the three components and also signal the release of stably assembled class I MHC molecules from the endoplasmic reticulum. A peptide-induced conformational change in HC has been demonstrated in cell lysates lacking beta 2-m to which synthetic peptides were added. Many features of this conformational change suggest that it may be physiologically relevant. In an attempt to study the peptide-induced conformational change in detail we have expressed a soluble, truncated form of the mouse H-2Db HC that contains only the peptide binding domains of the class I molecule. We have shown that this peptide-binding "platform" is relatively stable in physiological buffers and undergoes a conformational change that is detectable with antibodies, in response to synthetic peptides. We also show that the structural features of peptides that induce this conformational change in the platform are the same as those required to observe the conformational change in full-length HC. In this respect, therefore, the HC alpha 1 and alpha 2 domains, which together form the peptide binding site of class I MHC, are able to act independently of the rest of the molecule.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Glycoproteins encoded by the major histocompatibility complex (MHC)1 class I locus present peptide antigens to cytotoxic T lymphocytes. MHC class I molecules are transmembrane glycoproteins comprising a 45-kDa heavy chain (HC) with three extracellular domains (alpha 1, alpha 2, and alpha 3), noncovalently associated with beta 2-microglobulin (beta 2-m). These subunits are assembled in the endoplasmic reticulum (ER) in association with peptide epitopes that are either derived from the cytosol and delivered to the ER by the transporter associated with antigen processing, or generated in the ER itself (see Refs. 1 and 2 for reviews). Biochemical evidence shows that when an appropriate peptide binds to the HC:beta 2-m heterodimer, the complex is stabilized (3-6) and that, in this respect, peptide binding and class I assembly are linked phenomena in so much as the formation of an MHC class I-peptide complex can be seen as the assembly of a trimolecular complex of HC, beta 2-m, and peptide. The crystal structures of several MHC class I-peptide complexes have now been solved (reviewed in Ref. 7) and support this view, showing that the peptide ligand is deeply buried in the peptide-binding cleft formed by the alpha 1 and alpha 2 domains. In some cases, as much as 80% of the peptide is buried (8), and in two cases, a salt bridge forms over the bound peptide, between side chains of the alpha 1 and alpha 2 domains on either side of the cleft (9). It is difficult to envisage the diffusion of peptide in and out of this peptide-binding groove according to the classical image of a receptor-ligand interaction. Despite this, several attempts have been made to measure apparent equilibrium binding constants (10) (reviewed in Ref. 11). Also, dissociation rates have been measured for a variety of class I peptide complexes (10). In only one report have both kinetic constants (ka and kd) and thermodynamic constant (Ka) been measured in the same system, and this concluded that peptide-binding groove to which peptides bind is different from those from which they dissociate, the implication being that a conformational change occurs in the class I molecule upon peptide binding (12, 13). Such a conformational change has been observed indirectly in another system. In the absence of bound beta 2-m, free HC undergoes a conformational change that is detectable with antibodies (4, 14-16), and similarities in the structural requirements for this conformational change and those for stable peptide binding to HC:beta 2-m heterodimers suggest that this conformational change may be related or identical to that proposed for the HC:beta 2-m complex upon peptide binding (12, 17).

The relevance of such a conformational change is 2-fold. First, it provides a mechanism for the MHC class I binding site to bind a wide variety of peptide ligands with high affinity. It is more usual to observe receptor ligand interactions in which increased affinity is achieved only at the "expense" of greater specificity. Second, it provides a way in which the MHC class I molecule could signal its release from cofactors that are responsible for retaining incompletely assembled class I molecules in the ER.

As a means of producing MHC class I molecules in a soluble form that could allow a study of the peptide-induced conformational change using high resolution techniques, we chose to make only the portion of a class I molecule that contains the peptide binding site. Given the apparent structural independence of this peptide-binding platform, our strategy was to express residues 1-193 of the murine MHC class I molecule H2-Db in Escherichia coli and to renature it after solubilization in M urea. We show here the successful expression of this fragment and its ability to undergo a peptide-induced conformational change.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Vectors, Cells, and Antibodies-- pGMT7 is a 3.1-kilobase pair, pAR2067-based plasmid containing T7 promoter and terminators flanking a polylinker. E. coli strain BL21 DE(plys c)is ampr incorporates an isopropyl-1-thio-beta -D-galactopyranoside-inducible gene for T7 RNA polymerase, allowing high level, inducible expression of transfected DNA. The monoclonal antibodies B22.249 and 27-11-13 (4, 18) recognize the native conformation of the H2-Db heavy chain. Rabbit antiserum T18 was raised against D1-193b inclusion bodies. It recognizes epitopes on both Db and Kb heavy chains, which are lost upon beta 2-m binding but remain when heavy chains bind to peptides in the absence of beta 2-m.

Cloning and Expression of D1-270b and D1-193b-- Full-length Db heavy chain corresponding to nucleotides 1-874 (amino acids 1-270, D1-270b), and a fragment of the Db heavy chain corresponding to nucleotides 64-642 (amino acids 1-193, D1-193b) were amplified by a polymerase chain reaction from cDNA made from the Rausher-transformed thymoma cell line RMA-S (19, 20). The amplification incorporated a 5'-BamHI and 3'-HindIII recognition site and added nucleotides coding for four additional histidine residues at the C terminus of the fragment. The polymerase chain reaction products were cloned into the vector pGMT7, and sequenced by the dideoxy method before being transferred into the BL21 strain of E. coli for expression. Transformed bacteria were grown in L broth containing 100 µg/ml ampicillin to a density of 0.3-0.5 A600 units before adding 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside for 5 h to induce expression. Clones expressing over 20 mg of protein/ml of culture were isolated.

Purification and Refolding-- Both D1-270b and D1-193b were expressed as inclusion bodies and were purified from protein-expressing BL21 transformants as follows. A washed cell pellet was lysed in a minimum volume of 50 mM Tris, pH 8.0, 25% sucrose, 1% Nonidet P-40, 0.1% sodium deoxycholate, 5 mM EDTA, 2 mM dithiothreitol by sonication (14 µm on ice until the decrease in viscosity indicated that all DNA had been sheared). Following cell disruption, and the removal of cell debris by centrifugation, inclusion bodies were pelleted (5 min, 10,000 rpm). Inclusion bodies were then washed twice in 25 mM Tris, pH 8.4, 2 M NaCl, 2 M urea, 2 mM dithiothreitol, and resuspended in 25 mM Tris, pH 8.4, 8 M urea. D1-193b refolding was carried out by dilution into 100 mM Tris, pH 8.0, containing 2 mM EDTA, 0.5 M arginine as a stabilizer, and a redox couple consisting of 0.5 mM oxidized, 5 mM reduced glutathione to encourage appropriate formation of the intramolecular disulfide bond between Cys101 and Cys164. After concentration in a Centriprep C10 (Amicon), the renatured product was purified by gel filtration on a Superdex 75 FPLC column equilibrated with TBS, using FPLC (Pharmacia Biotech Inc.). Fractions containing soluble, monomeric D1-193b were collected, pooled, and concentrated further by membrane filtration. Protein concentrations were determined by the method of Lowry.

Small, experimental refolding reactions were carried out for analysis by enzyme-linked immunosorbent assay in which 50 µg of D1-193b or D1-270b inclusion bodies resuspended in 10 µl of 8 M urea were refolded in 2 ml of refolding buffer containing different concentrations of peptide for 48 h at 4 °C. Where appropriate, the refolding buffer also contained 1.36 µM recombinant beta 2-m.

Immunoprecipitation-- 1-ml samples of D1-193b containing 2-10 µg of protein were incubated at 4 °C with 50 µM of either the H2-Db-restricted influenza A nucleoprotein epitope residues 366-374 (ASNENMDAM), or the H2-Db and H2-Kb-restricted Sendai virus nucleoprotein epitope residues 324-332 (FAPGNYPAL) for 1 h. D1-193b was then immunoprecipitated by adding the antibodies (10 µg/ml for the monoclonal antibodies, 1/500 dilution for the antiserum), incubating for 90 min at 4 °C, then precipitating immune complexes with 50 µl of 10% w/v protein A immobilized on Sepharose 4B (Sigma) for 30 min. The precipitates were washed four times in TBS, then analyzed by SDS-polyacrylamide gel electrophoresis.

Enzyme-linked Immunosorbent Assay-- 96 flat-bottomed well, UPVC plates were coated overnight with 200 µl of 1 µM soluble D1-193b-ASNENMDAM complex in phosphate-buffered saline. Excess sites were blocked with 2% bovine serum albumin in phosphate-buffered saline at 4 °C for 2 h. 100 µl of unpurified refolding mix were preincubated at 4 °C for 2 h with BB7.2 at a final concentration of 250 ng/ml in phosphate-buffered saline, 2% bovine serum albumin, after which time the whole mixture was transferred to wells of the coated plate and incubated at 4 °C for 1 h. After removal of the mixture, the wells were washed three times in 200 µl TBS, 0.5% (v/v) Tween 20, 0.4% bovine serum albumin, and 200 µl of alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma) were added (1/1000 dilution in TBS) and incubated for 1 h at room temperature. Following a further three washes, 200 µl of substrate containing 1 mg/ml p-nitrophenyl phosphate in 200 µl of TBS were then added to each well and incubated at 37 °C for 20 min before reading A405 on a Titertek Multiskan plate reader.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning, Expression, and Purification of the H2-Db Peptide-binding Platform D1-193b-- The 193-amino acid fragment amplified from cDNA lacks the signal sequence (residues 1-22), most of the alpha 3 domain, the transmembrane, and cytoplasmic domains and is called D1-193b. In addition, the C terminus of the fragment was tagged with four additional histidine residues, which along with the two naturally occurring histidine residues at the C terminus of this fragment, could be used to purify the protein by nickel chelation chromatography. We chose this fragment rather than a shorter one encompassing only the alpha 1 and alpha 2 domains in order to preserve a conserved salt bridge between Arg181 and Asp183, which we felt could be important in stabilizing the platform. Previous attempts to express slightly shorter fragments of other alleles have been unsuccessful in producing a soluble antigen binding platform.2

Following induction of expression for 5 h, most transformants expressed D1-193b as inclusion bodies. Clones expressing greater than 20 mg of protein per liter of culture were isolated and expanded. Fig. 1 (lane a) shows that the crude inclusion body preparation contained a polypeptide of a size corresponding to D1-193b (20 kDa) in over 70% abundance. Following washing, greater than 95% of the urea-soluble material was D1-193b. Refolding, with the replacement of 8 M urea with a stabilizing salt (0.5 M L-arginine), allowed the recovery of around 50% of the input polypeptide in a soluble form. This contained a mixture of products comprising approximately 10% disulfide bonded trimer, 40% disulfide-bonded dimers, and 50% monomer as determined by reducing and nonreducing SDS-polyacrylamide gel electrophoresis, with very few high order soluble aggregates (data not shown). Thus, around 5 mg of soluble monomer per liter of culture, was recoverable by gel filtration in TBS, 0.5 M arginine. This was stable at around 1 mg/ml for at least 4 weeks. In the absence of the stabilizing solute, D1-193b was slightly less stable and tended to form dimers and trimers when stored at around 0.5 mg/ml at 4 °C for 4 weeks. Routinely, renatured D1-193b was purified and simultaneously transferred into TBS by FPLC gel filtration. A typical chromatogram is shown in Fig. 2. When 50 µM peptide was included in the renaturation, the yield of monomeric D1-193b increased slightly when analyzed by gel filtration, with a corresponding decrease in the yield of dimers and trimers (Fig. 2). In contrast to D1-193b, full-length heavy chain (D1-270b) aggregates completely in the absence of any added peptide during the refolding reaction. This indicates that the formation of low order soluble aggregates is promoted by the presence of the alpha 3 domain.


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Fig. 1.   Expression of D1-193bD in E. coli and its purification from inclusion bodies. Samples of a, pelleted inclusion bodies; b, supernatant; c, detergent wash; d, urea-insoluble material; and e, urea-soluble material isolated from these inclusion bodies were resuspended in 1% SDS, boiled, and analyzed by fractionation in a 10% polyacrylamide gel.


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Fig. 2.   Purification of monomeric D1-193b by FPLC gel filtration. After refolding in the presence (thin line) and absence (thick line) of 20 µM ASNENMDAM, concentrated D1-193b was fractionated on a Superdex 75, FPLC column. The column had previously been calibrated with standard proteins of 158, 44, and 17 kDa (marked). Fractions corresponding to monomeric D1-193b were pooled and concentrated.

Biological Activity of D1-193b-- We have previously shown that, in the absence of beta 2-m, short peptides that specifically bind to Db can induce a conformational change in the heavy chain in cell lysates (4, 14, 15). We therefore investigated the ability of Db-binding peptides to induce a conformational change in D1-193b in solution. In TBS (and TBS/arginine), the soluble monomer was unreactive with two monoclonal antibodies that recognize epitopes present in the native conformation of H2 Db alpha 1 and alpha 2 domains (B22 and 27-11-13). It was, however, recognized by antiserum T18 which was raised to D1-193b (see "Experimental Procedures"). Fig. 3 shows that, when either of the Db binding peptides (FAPGNYPAL or ASNENMDAM) were added to a final concentration of 50 µM, the conformation-sensitive epitope is recovered, and D1-193b could now be recognized by mAb B22.249. This is more readily observed for the latter peptide, which has a higher apparent Ka for H2-Db,3 and strongly suggests that the peptides induce a conformational change in D1-193b upon binding. No such conformational change is observed when a control peptide (the H-2Kb-binding peptide SIINFEKL) was added to a concentration of 100 µM (Fig. 3, c and d). These results are consistent with those obtained for full-length and truncated heavy chains studied in detergent lysates of mammalian cells that do not express beta 2-m (4, 14, 15). These chains, unlike D1-193b, are N-glycosylated. Thus the absence of N-linked carbohydrate does not appear to influence the ability of the alpha 1 and alpha 2 domains to undergo this conformational change in response to optimal peptides.


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Fig. 3.   D1-193b undergoes a conformational change in response to specific peptides. Samples of D1-193b which had been incubated with the Kb binding peptide SIINFEKL (a-d), the Db- and Kb-binding peptide FAPGNYPAL (e-h), or the Db-binding peptide ASNENMDAM (i-l) were immunoprecipitated with preimmune rabbit serum (a, e, and i), antiserum T18 (b, f, and j), the Db-binding mAb B22 (c, g, and k) or the Kb-binding mAb Y3 (d, h, and l). Proteins were detected by Coomassie Blue staining following analysis of the immunoprecipitates by SDS-PAGE. D1-193b runs below Ig light chains (LC), which are visible for the monoclonal antibodies but not for the polyclonal antisera, presumably due to LC heterogeneity. Ig heavy chains (HC) are also visible.

In an attempt to quantify the ability of peptide to induce the conformational change in both D1-193b and D1-270b in solution, small scale refolding reactions were initiated in the presence of different concentrations of FAPGNYPAL (WT). The recovery of mAb B22-reactive D1-193b and D1-270b was then quantitated by enzyme-linked immunosorbent assay. mAb B22 was mixed with crude, unfractionated refolding reactions, and the mixture was applied to 96-well plastic plates coated with previously refolded, purified complexes of D1-270b + beta 2-m + ASNENMDAM. Soluble, folded Db molecules in the folding mix will block the B22 antibody, which is at limiting concentration, from interacting with the immobilized complexes, reducing the amount of antibody that can bind to the plate. Neither the soluble aggregates present in the refolding mix nor free beta 2-m or peptide can bind to mAb B22 (data not shown). The percentage maximum inhibition (compared with a folding to which no peptide has been added) is therefore proportional to the yield of correctly folded, B22-reactive material in each in vitro refolding reaction. Fig. 4a shows that, when beta 2-m is included in the folding reaction with D1-270b, the recovery of B22-reactive molecules is much more efficient than for either D1-193b or D1-270b in the absence of beta 2-m, requiring approximately 1000-fold less peptide to achieve the same yield of folded material. A similar observation has been made for the binding of peptides to full-length Db heavy chains in cell lysates in the presence and absence of beta 2-m (4, 14, 15). The presence of beta 2-m did not affect the folding of D1-193b (data not shown). These experiments indicate that the alpha 3 domain appears to contribute little to the ability of heavy chains to bind peptides when beta 2-m is not present, but increases the binding efficiency severalfold when beta 2-m is present, presumably by contributing to the beta 2-m binding site, allowing beta 2-m to stabilize the peptide-binding groove. These results are consistent with similar observations made for a Kb molecule with a deleted alpha 3 domain expressed in beta 2-m-negative cells (21).


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Fig. 4.   Quantitative refolding of D1-270b and D1-193b in the presence of increasing amounts of WT peptide FAPGNYPAL. a, the effect of changing anchor residues at positions 5 (A5, FAPGAYPAL) and 5 and 9 (A5A9, FAPGAYPAA) on the recovery of refolded HC:beta 2-m complexes from refolding reactions containing D1-270b and beta 2-m is shown in b. The same effect was measured for D1-270b and D1-193b in the absence of beta 2-m (c).

The influence of the anchor residues (asparagine 5 and leucine 9) on the ability of a peptide to induce the conformational change was assessed by initiating folding reactions in the presence of analogues of the WT peptide carrying alanine substitutions at positions 5 and 9 (Ala-5, FAPGAYPAL; and Ala-5 to Ala-9, FAPGAYPAA). Fig. 4b shows that for D1-270b in the presence of beta 2-m, changing asparagine 5 to alanine had a relatively small effect compared with WT, reducing the efficiency with which peptide induces folding by less than 4-fold. Changing leucine 9 to alanine resulted in a further 3-fold reduction in folding. The asparagine 5 to alanine substitution had a more dramatic effect on the folding of D1-193b or D1-270b in the absence of beta 2-m (Fig. 4c), reducing the recovery of folded molecules by almost 100-fold, and when leucine 9 was also changed to alanine no significant folding was seen within the range of peptide concentrations used (up to 760 mM, equivalent to a 1000-fold molar excess). There is therefore a more stringent requirement for both peptide anchor residues for the incorporation of peptide into refolded Db in the absence of beta 2-m.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

This is the first description of a soluble fragment of an MHC class I molecule comprising the two N-terminal domains of the protein that form the antigenic peptide-binding site. In the absence of peptides, this 193-amino acid fragment is reasonably stable in physiological buffers, but the two domains exist in a non-native conformation as judged by their inability to be recognized by monoclonal antibodies raised against the native whole molecule. A similar situation is seen for full-length heavy chains synthesized in mammalian cells that lack beta 2-m. Here, although the immunolglobulin-like alpha 3 domain appears to be in a native conformation, the alpha 1 and alpha 2 domains do not. In detergent lysates, these molecules undergo a conformational change when they bind to antigenic peptides in the absence of beta 2-m (4, 14-16), such that the alpha 1 and alpha 2 domains acquire epitopes present in the native structure. We have also suggested that peptides bind to the non-native conformation of heavy chain and in doing so, initiate the conformational change (15). This conformational change does not require the presence of the alpha 3 domain (14), an observation which led us to speculate that it might be possible to observe the same conformational change in the isolated peptide-binding platform formed by the alpha 1 and alpha 2 domains. Our results show that this is indeed the case and open up the possibility of studying the conformational change by conventional biophysical techniques. Indeed, D1-193b is of a size which is compatible with two- dimensional NMR spectral analysis (22, 23).

We have suggested that the conformational change which we observe for free heavy chain, and now the isolated peptide-binding platform, may be related to a conformational change, which is thought to occur in the heavy chain when peptides bind to the assembled HC:beta 2-m heterodimer in vivo. This has been observed indirectly by fluorescence transfer between two MHC class I-bound mAb (17), and by immunoprecipitation of peptide receptive and peptide-bound H2-Ld molecules with mAb which discriminate between the two forms (24). A more persuasive, but no less indirect, indication of the conformational change arose from a measurement of the kinetic and thermodynamic binding constants of H2-Db for Db-binding peptides (12, 13). This study showed that, for an N-terminally extended peptide ligand, the measured association rate constant corresponded to that predicted from a simple one-step binding model in which Ka = ka/kd. However, for the optimal length peptide, the measured association rate is two orders of magnitude higher than that predicted from the kd and Ka. This led to the proposal that a conformational change in the class I molecule was responsible for the observed the mismatched kinetics of peptide binding, and that only peptides of the optimum length can bring it about. It is interesting to note that the conformational change seen in free heavy chains and D1-193b is also observed only in response to optimal length peptides, and that these appear to induce the conformational change rather than simply stabilize the native conformer preferentially (15).

Since the solution of the first MHC class I structure (25), immunologists and structural biologists have been puzzled by the observation that the peptide ligand is so deeply buried in the peptide-binding groove as to be considered part of the MHC class I structure, and have found it difficult to visualize how peptides might diffuse into the binding site as it appears in the crystal structures. This is made all the more difficult when the relatively rapid association rates that have been measured are taken into account. A peptide-induced conformational change from a more "open" or "receptive" peptide-binding groove to the "closed" structure seen by x-ray crystallography would be consistent with these observations. The exact molecular dynamics that constitute this conformational change are entirely unknown at the present time, but a testable model has recently been proposed that involves a movement in the short alpha  helix of the alpha 2 domain (26).

A peptide-induced conformational change in the MHC class I molecule might also explain why newly assembled HC:beta 2-m heterodimers are retained in the endoplasmic reticulum until they become loaded with peptides of an appropriate length and sequence. It is possible that a cofactor in the ER with an ER-retention signal is able to bind to peptide-receptive but not peptide-loaded MHC class I molecules and that the ability to discriminate between the two forms is due to a peptide-induced conformational change. Indeed, in a nonphysiological experimental system, invariant chain (a cofactor molecule that is normally involved in the biogenesis of MHC class II molecules and not class I) has been shown to bind to H2-Db in a peptide-sensitive manner (27) in vivo. Other, physiologically relevant candidates are the calcium-binding chaperones calnexin and calreticulin (28, 29) and the transporter associated with antigen processing (30, 31).

The production of D1-193b and the demonstration that in the presence of specific optimal peptides it is recognized by conformation-sensitive mAb, provides, for the first time, a means of studying the peptide-induced conformational change by direct biophysical methods.

    FOOTNOTES

* This work was supported in part by the Wellcome Trust and the Nuffield Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Wellcome Senior Fellow in Basic Biomedical Science. To whom correspondence should be addressed. Tel.: 01865 221949; Fax: 01865 22901.

1 The abbreviations used are: MHC, major histocompatibility complex; HC, heavy chain; beta 2-m, beta 2-microglobulin; ER, endoplasmic reticulum; FPLC, fast protein liquid chromatography; TBC, Tris-buffered saline; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; WT, wild type.

2 P. Bjorkman, personal communication.

3 T. Elliott, unpublished observation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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