MXPA01002674A - A method of producing a functional immunoglobulin superfamily protein. - Google Patents

Abstract

The present invention relates to a process of producing a functional immunoglobulin superfamily protein, which has at least one disulphide bond when functional, the process comprising the steps of providing a bacterial cell comprising a gene coding for the protein, the gene is expressible in said cell, cultivating the cell under conditions where the gene is expressed, isolating the protein from the cell without reducing it, and subjecting the isolated protein to a folding treatment. Preferably, the immunoglobulin superfamily protein is selected from the group consisting of antibodies, immunoglobulin variable (V) regions, immunoglobulin constant (C) regions, immunoglobulin light chains, immunoglobulin heavy chains, CD1, CD2, CD3, Class I and Class II histocompability molecules, beta2microglobulin (beta2m), lymphocyte function associated antigen-3 (LFA-3) and FcgammaRIII, CD7, CD8, Thy-1 and Tp44 (CD28), T cell receptor, CD4, polyimmunoglobulin receptor, neuronal cell adhesion molecule (NCAM), myelin associated glycoprotein (MAG), P myelin protein, carcino-embryonic antigen (CEA), platelet derived growth factor receptor (PDGFR), colony stimulating factor-1 receptor, alphabeta-glycoprotein, ICAM (intercellular adhesion molecule), platelet and interleukins. Important embodiments of the invention is a stable peptide free MHC protein obtainable by a process of the invention and a kit comprising a MHC class I heavy chain and a beta2m allowing the recipient to produce and measure or detect a functional MHC class I protein to which a peptide, which is capable of binding to said MHC class I protein, can be added leading to the generation of a functional MHC class I protein.

Description

A MIETODE TO PRODUCE A FUNCTIONAL IMMUNOGLOBULA SUPERFAMILY PROTEIN Brief Description of the Invention • 5 The specificity and reactivity of the human immune system is governed by the MHC molecules (in human HLA). The function of HLA is to select and present antigenic peptides for immune T cells. It can be said that the • The immune system sees the world through the eyes of the MHC, and that any rational approach to immune manipulations must take the MHC into consideration. Such rational approaches have many scientific, practical or clinical uses. To exploit these potentials, a method is desired that generates high purity recombinant MHC molecules in a novel, simple, robust and inexpensive manner. These 20 recombinant molecules are functionally fully active as peptide linkers and T cell stimulators. They can be generated in two distinct ways: a) as a fully mature peptide filled portion, which is extremely stable and stimulates the cell REF .: 128222 T, and b) as a partially mature peptide-free portion ("empty" MHC molecules), which is reasonably stable and readily receptive to the peptide. It will be noted that the last result • corrects the current misconception that "empty" MHC molecules are not existent, or at least extremely unstable.

All other production methods of the 10 recombinant or non-recombinant MHC known for • used are much more uncomfortable and / or generate products of limited efficiency and purity. The complexity and poor stability of the MHC molecules make them very difficult to generate pure peptide receptors and MHC molecules and, consequently, it is very difficult to generate pure and predetermined peptide-MHC complexes. As natural molecules from natural sources, the production of MHC molecules is tedious and of low performance. Additionally, these can only be purified by uncomfortable methods leading to preparations where the MCH is previously occupied by a large range of different peptides and is sometimes contaminated by other MHC haplotypes with the net result being highly contaminated preparations.

The composition problems to generate • MHC molecules is the extreme polymorphism of the MHC site. In the human population, there are more than 400 different HLA-A, HLA-B and HLA-C alleles, and more than 200 HLA-D alleles. This diversity has an immunological purpose, but it is an obstacle 10 practical for the production of MHC because it • they need many different MCHs to generate and optimize individually, validate, characterize, store, etc. 15 A recombinant expression system can have several advantages. Potentially high performance, easy purification scheme, and molecules will be marked • for homogeneity with a particular peptide. 20 The biggest obstacle to such an approach, however, is that the properly retained MHC structure is a rather complicated structure consisting of three components (a heavy chain, a light chain (bet a2 -microglobulin, ß2m) and a Peptide). The complete stability of the MHC is only acquired when the three complete components are together. In particular, the heavy chain is extremely unstable in the absence of the other two components. In this way, it is difficult to • producing, handling and storing the heavy chain in isolation without the ß2m and the peptide, and it is therefore difficult to generate MHC molecules, which are readily available to bind in any peptide of the chosen experiments. Due to the 10 limited stability, MHC molecules isolated • they quickly add up and are less noticeable for extremely poor final returns.

This patent application describes a General approach to generate recombinant MHC molecules in large quantities of any of the specifically desired alleles. The method is also done to • MHC molecules in a first time completely 20 empty, even reasonably stable, and the results show that the binding characteristics of these empty molecules deviate from those obtained with MHC generated in the past. It is a reasonable expectation that these molecules 25 empty are more relevant than the physiologically linked MHC since they reflect a n ovo bond, while previously used MHC molecules have more artificial reflections of exchange reactions • 5 in addition to the quality of the improved MHC, the production scheme Novelty is easy and robust, easily adaptable to most (probably all) of the MHC molecules and has a high yield of pure and fully functional MHC molecules. These recombinant molecules are extremely potent as can be detected from the peptide binding activity of at least 2-4 ng of heavy chain MHC, and the binding capacity to T cell can be detected which is less than 150 ng peptide. / MHC. These are extremely active with an affinity that is similar to that measured for naturally occurring molecules, a much faster • association ratio, and 20 fully available for binding (this is without endogenous peptides). They also have the ability to store these molecules and maintain their activity for many months. The implications are both analytical and therapeutic.

• A- ^ fltfMiÉttb Finally the methods described are successful when applied to other molecules (non-MHC) such as those of the CD3 complex. In fact, the described methods will be used in any protein production scheme (whether it is recombinant or not, if it is in prokaryotes or eukaryotes) where the protein at some point during production is solvated by denaturation, or is released (the purpose should be to dissolve aggregates 10 protein, to purify the protein etc.) • needing a last step of renaturation / refolding. In this way, the method can have a very large field of application. In particular, it is speculated that they can 15 be useful for all members of the immunoglobulin superfamily including antibodies, MHC receptors and T cell. It can be useful for the production of all molecules • that contain at least one cysteine. Background of the Invention.

The immune system can be observed as one of the natural bioinformatic systems. East 25 evaluates any substance that enters the internal environment, determines its nature and decides what type of action to take against it. Proteins and peptides are the most important means to obtain and convert such immune information. From this point of view, MHC molecules are at the center of the immune system. The MHC molecules show the metabolism of the complete protein for peptide information and make this information available to the central recognition unit of the immune system, the T cell. The current patent application refers to a larger order for generate suitable methods to determine and predict the function of the MHC (a complete mapping of all reactive human MHCs). Combining the genome databases of growth of primary protein sequences of humans and parasites with the • precise knowledge of whether this immune molecule 20 handles peptide information that leads to new and powerful strategies of epitope prediction. This improves the possibilities to direct and make more efficient the immune manipulations. The ability to generate recombinant MHC molecules 25 allows this MHC / HLA mapping project to large JÜJÍJ - ^ "'« "~ - *" S ~ scale, and also has practical, clinical and scientific uses of immediate commercial interest as described below.

• Introduction The purpose of the immune system is to protect our body against organisms of microbes (eg bacteria, viruses, 10 parasites) and maybe against cancer. Virtually any threat can be eliminated or neutralized by the immune system. To administer such power, the immune system must know that it attacks it, and that it does not attack it; Ideally, the foreign matter must be removed while the body itself must remain without prejudice. The true mark of the immune system is therefore a specific one, that is, the ability to discriminate between several objectives 20 and in particular to distinguish between what is of the / what is not. The specific immune system consists of a large number of cells, or lymphocytes, with a greater subdivision into B and T cells that represent humoral responses 25 (antibody) and cell phones, respectively. Both cells use receptors, which in their genome encode many impulses and pieces allowing a recombinatory diversity of the enormous receptor. Each of the B or T cells carries one, and only one, of these receptors and can recognize a very thin part of the universe. All the combined lymphocytes, however, can recognize the universe. The general specificity of the immune system is generated, regulated and coordinated through the control processes of the individual lymphocytes. Withdrawing, or inactivating, a lymphocyte clone removes the corresponding specificity of the repertoire. The activation and propagation of a lymphocyte clone increases the corresponding specificity, and allows the immune system to rapidly respond strongly if it is exposed to the same antigen again.

Cells B and T specifities B and T cells use completely different mechanisms to recognize their targets. B cells recognize soluble antigens, and since they can secrete their receptors as antibodies, they can potentially interact with an antigen through the fluid phase of the extracellular space. In a marked contrast, the T-cell receptor is always a bound membrane and only recognizes an antigen, which occurs in the membrane, thus being called a cell present in the antigen (APC). In other words, recognition of the T cell involves a direct physical interaction between the two cells, a T cell and an APC. B and T cells also differ with respect to what they can recognize. B cells can recognize organic substances of almost any type, while T cells only recognize proteins (as a biological target, proteins that are particularly important since these constitute the structure and functional bases of all life). B cells recognize the three-dimensional structure of proteins as illustrated by their ability to distinguish between native and denatularized proteins. In contrast, T cells can not distinguish between native and denatured proteins. Previously, this led to the idea that T cells recognized altered proteins. It is now known that this is true and that T cells only recognize antigenic peptides presented in association with MHC molecules on the surface of APCs. In general, cytotoxic T cells recognize • 5 short peptides (8-11 mers) whose amino and carboxy termini are deeply embedded with the MHC (that is, the long peptide is restricted). In contrast, helper T cells tend to recognize long peptides (13-30 mers or more large 10) with terminal amino and carboxy termini extended outside the MHC (ie the long peptide is not restricted).

Immune Responses and Restriction of MHC 15 T cells are of particular importance for the induction of immune responses since they determine the reactivity and specificity of the entire immune system, including B cells. Therefore, it is appropriate to focus our attention on this cell. T cells can only recognize an < given antigen, when it occurs in the context of a particular MHC molecule. These are "educated" during ontogeneity and are also activated during the first priming in processes that ^^ tm m are designed to develop T cells that carry specific receptors for a particular antigen-MHC combination. These T cells can only subsequently recognize the same antigen exactly the same as the MHC combination. This phenomenon is known as "MHC restriction". Another immune phenomenon, which "responds to the state", is also determined by the MHC. Individuals of an MHC aplotype will respond to some antigens, and others will not. Other individuals with other MHC aplotypes will respond differently. These two phenomena are of obvious importance for any rational immune manipulation. As mentioned, it is now known that both are controlled by MHC molecules. These molecules, which have a development specifically for the purpose of an antigen presentation. Our current knowledge of antigen presentation can be summarized as follows. First, the foreign substance, the antigen, is taken up by the APCs. An intracellular well of antigenic peptides is generated through the protiolytic fragmentation of all protein antigens available in the cell. This well of peptides are offered for the individual's MHC molecules and sample of • - ***** > ** u conformity to length and consequence; some are linked, while others are ignored (the MHC is said to develop "a determining selection." Subsequently, the MHC molecules protect the selected peptides against further degradation, transport them to the surface of the APC and expose them to a scrutiny of the T cell MHC and Polymorphism There are two subtypes of MHC, MHC class I and class II. These subtypes correspond to two T lymphocyte subunits: 1) CD8 + cytotoxic T cells, which usually recognize peptides presented by MHC class I molecules, and eliminate T cells from infected or mutated cells, and 2) CD4 + helper T cells, which recognize usually peptides presented by MHC class II molecules, and regulate the responses of other cells of the immune system. Class I MHC consists of 43,000 MW transmembrane glyc protein (α chain) non-covalently associated with a non-glycosylated 12,000 MW protein (the β chain also known as β2-microglobulin). The MHC The class II has a structure similar to the MHC class I, although the distribution of the domain is different. Class II consists of two non-covalently associated transmembrane glycoproteins of approximately 34,000 and 29,000 MW. The detailed structure of the MHC class I and class II molecules is provided at the level of X-ray crystallography (Bjórkman et al., 1987). The most interesting part of the MHC structure is the "upper" part that shows a single ribbed peptide bond consisting of two alpha elixirs that form the groove walls and eight beta-planted nuclei forming the grooved floor.

The MHC is extremely polymorphic, that is, there are many different versions in the population (a-lelos, allotypes), but each one individually inherits only one or two of these (one from the father and one from the mother). It is also polygenic, that is, there are several coding sites of the MHC in the genome that are allowed for the simultaneous expression of several isotypes. Importantly, most polymorphic residues point towards the peptide ^^^^^^^ linked to the groove affecting its size, shape and functionality (Matsumura et al., 1992). The peptide-MHC interactions are specified, broadly, allowing the binding of many • 5 peptides not related to each of the MHC allotypes (Buus et al., 1987). The polymorphism dictates the specificity of the bound peptide and the biological consequence of this, because each of these individuals in the population educates and forms a repertoire of unique T cells.

Generation of specificity of the MHC.

Structurally, the peptide binding site of the MHC forms a groove that can be subdivided into several packets, A through F. Most of the MHC peptide bond energy involves the main chain atoms of the MHC.

Linked peptide (including the term for the MHC class I); characteristics that are common for all peptides (Matsumura et al., 1992). Only the minority of linked energy involves chain atoms 25 side of peptide, however, these interactions are considered to explain the specificity of the MHC. This mechanism explains why the MHC executes a broad specificity, even with high affinity of the peptide bond. Functionally, the MHC makes the broad peptide bond specifically through the recognition of its "motifs" (Sette et al., 1987). A motif represents important structural requirements needed to bind the peptide of 10 such as the presence and space of • particular amino acids at anchor positions. Considerable interest has been focused on the understanding that specific MHC and motives are generated, and on characterizing the specificity of 15 several MHC molecules. One of the latest successes of these efforts is the ability to predict the peptide bond. Following the polymorphism of the MHC, (since both are provided • structurally as well as functionally) each of 20 the allotypes MHC has its own characteristics of specificity. Until now these specifics can only be described experimentally.

Description and Prediction of the Specificity of the MHC Two fundamentally different but complementary approaches are currently used to determine the specificity of the MHC peptide link. One approach consists of sequencing the peptide already bound in the MHC molecules of a given allotype (Buus et al., 1988; Falk et al., 1991), whereas the other approach consists of examining that the peptide binds to the given MHC ( Buus et al., 1986; Olsen et al., 1994). Both approaches have advantages and disadvantages. The sequencing method deals with naturally-processed MHC peptide complexes, however, important residues are arbitrarily assigned against less important versus unimportant ones and no residues that interact negatively are identified. This is most appropriate to identify the majority of the dominant residues that interact in a positive way. The last approach, the direct link method, is quantitative and allows a comparison of binders against non-binders. Residues that interact both positively and negatively can be identified quantitatively. It is perhaps not surprising that the direct link method produces a better predictive capacity (around 70% of the predicted peptides to bind), than the sequencing method (around 30% success) (Kast and Cola ooradores, 1994). It has been shown that acute predictions of peptide binding require that the fine specificity of the MHC in question be known in detail (sometimes called extended motifs) (Parket et al., 1994; Stryhn et al., 1996). However, to obtain such detailed motives is a very intensive and highly developed work. Currently, to determine the fine specificity of each of the MHC molecules of interesting long panels of peptides that influence particular sequences or motifs, it is analyzed routinely (Parket et al., 1994; Rupert and Cola oradores, 1993). An approach based on a collection of peptides has recently been developed, yielding a correct, uninfluenced and quantitative description of all functionally important MHC linked residues (Stryhn et al., 1996). This is universal since many different MHC molecules can be targeted with the same set of peptide libraries and evaluated to test their binding that they can develop for each of the MHC molecules. Conveniently, this approach means a reduction in the experimental steps and the subsequent data handled and can therefore facilitate the complete mapping of all the MHC class I speci fi cations. This approach based on the collection of uninfluenced peptides does not lead either. to improve peptide bond predictions (Stryhn et al., 1996). Success in predicting the algorithm implies that the MHC class I link can be widely observed as the result of a formation of sub-specifics that act independently.

Generation of recombinant MHC molecules It has been shown by others (Parket and Wiley, 1989) that the bacterium can be a production vehicle for recombinant MHG molecules. However, packaged in inclusion bodies within the bacterium, these molecules have not been appropriate for binding peptides. Strategies involving complete denaturation and reduction of these inclusion bodies have been used to extract and solubilize the recombinant MHC molecules (Parket et al., 1992; Parket et al., 1992; Parket and Wiley, 1990). This necessarily leads to using relegation procedures in the presence of reducing / oxidizing agents, for example glutathione (GSH / GSSG). Other components such as L-arginine have been added to prevent aggregation and unfolding. However, the folding in vi t ro faces a major problem in the generation of properly formed di-sulfide bridges. The MHC class I heavy chains contain 4 (in some molecules 5) cysteines. There are several possibilities for disparate disulfide bridges during such retracted. The overall performance of MHC class I using this approach is reported to be very low (around 10-20%), and of completely slow kinetics (Garboczi et al., 1992).

Detailed Description of the Invention The present invention relates to a process that is invented as an object of solving the problem of having functional immunoglobulin superfamily proteins expressed in aggregates such as inclusion bodies. In the process of the invention, the functional protein may consist of several subunits of protein that are generated in the same cell or in different cells. In the latter case, the functional proteins, which may very well be two different types of proteins, for example, a heavy chain of an MHC class I protein and a β2 microglobulin, may be combined at the time of folding or later.

In one embodiment, the invention relates to a process for producing proteins of the functional immunoglobulin superfamily, which have at least one disulfide bond when they are functional, the process comprises the steps of (i) providing a bacterial cell comprising a gene encoding the protein, the gene is expressible in said cell, (ii) culturing the cell under conditions where the gene is expressed, (iii) isolate the protein from the cell under • 5 conditions, which do not change the disulfide bonds generated by the cell, and, optionally, purify the protein, (iv) subjecting the isolated protein to a folding treatment.

When the term "the" or "an" is used in the present specification and the claims, this means that it is one or more, that is at least 15 one.

By the term "a functional protein" means a protein of the immunoglobulin superfamily that is capable of running at least One of the functions attributed to said protein is at least one substantial degree, for example, an evaluation by a multiple test. By way of example, "a MHC class I functional protein" is defined as a protein comprising a 25 heavy chain, one light chain (b2m) and one The peptide can be truncated in order to render it soluble in an aqueous solution The peptide is a peptide that can bind to the MHC protein in question Such peptides can be found by, for example, the method of direct linkage described in Buus and Cola speakers 1986, and Olsen and colleagues 1994.

By "a MHC class II functional protein" means a protein comprising a complex of two heavy chains (a chain a and a chain β) and a peptide. The heavy chains can be truncated in order to make the complex soluble in an aqueous solution. The peptide is a peptide that can bind to the MHC protein in question. Such peptides can be found by means of, for example, the direct binding method described in Buus et al. 1986 and Olsen et al. 1994.

Particularly preferred embodiments of the invention are processes wherein the MHC protein is a MHC class I protein selected from the group consisting of a heavy chain, a past chain combined with a β2m, • «• MttJ¡ÜA? a mature functional protein MHC class I, or an MHC class II protein selected from the group consisting of an a / β dimer and a / b dimer with a peptide. An important aspect of the invention is a process wherein the MHC protein produced is obtained as a peptide-free MHC protein.

By "a peptide-free class I MHC protein" means a protein comprising a heavy chain associated with a light chain (b2m) but without peptide. Such a protein can also be called an "empty" class I MHC protein.

By "a MHC class II protein free of peptides" means a protein comprising a heavy chain complex but without a peptide. Such a protein can also be called an "empty" class II MHC protein.

The present invention is exemplified with reference to MHC class I proteins, but it is conceived that it may be possible in a similar manner to generate all the proteins of the immunoglobulin superfamily (these are by definition linked to disulfide), that is, a protein selected from the group consisting of antibodies, variable immunoglobulin regions (V), constant immunoglobulin regions (C), immunoglobulin light chains, immunoglobulin heavy chains, CD1, CD2, CD3, histocompatible class I and class II molecules, β2 -my croglobulin (ß2m), lymphocyte function associated with antigen-3 (LFA-3) and Fc? RIII, CD7, 10 CD8, Thy-1 and Tp44 (CD28), T cell receptor, CD4, polyimmunoglobulin receptor, neuronal cell adhesion molecule (NCAM), myelin-associated glycoprotein (MAG), myelin P protein, antigen ionic carcinoembr 15 (CEA), platelet-grade growth factor receptor (PDGFR), colony factor I stimulating receptor, a-β-glycoprotein, ICAM (molecular adhesion molecule), platelets and interleukins. The The present inventors have already provided data with respect to several MHC class I ß2 molecules - my croglobulin, MHC class II molecules, CD3 T cell receptors with gamma and epsilon chains. 25 The cloning of cDNA encoding the different proteins of interest is followed by standard procedures, for example, as described in Molecular Cloning (Sambrook, Fritsch • 5 and Maniatis, Cold Spring Harbor Press, 1989). Briefly, the cDNA is synthesized from appropriate cell lines using commercial cDNA synthesis kits (in this case from Pharmacia). For human work, the cells are derived from 10 part., Of HLA-transformed human EBV cell lines of the 12th International Histocompst ibility Workshop Cell Lines Panel Datábase ("HLA: Genetic diversity of HLA. Functional and Medical Implantation", Ed. Dominique 15 Charron, EDK press, 1997, or see http: // www. icnet.uk/axp/tia/marsh/ihw.htral). For HLA-A * 0201 an appropriate cell line will be IHW 9012. The nucleotide sequence corresponding to the desired MHC / HLA molecule can be 20 found at http://www.anthonynolan.com/HIG/index.html, or at http://www.ncbi.nlm.nih.gov. Using this sequence information, the oligonucleotide primers can be designated to amplify 25 by the polymerase reaction chain of the encoded region, encompassing amino acids 1-274 of the mature MHC / HLA molecule of the appropriate cDNA. A set of relevant front and rear primer is placed for the purpose of amplifying HIL-A * 0201 and inserting it into the Ncol and HindIII restriction sites of the pET28a expression vector (Nova-gen, see http: //www.novagen. com / vectfram.html). The ligated product is transformed into the TOP10F 'bacterium and selected for kanamycin resistance overnight. Several clones are taken and their plasmids are prepared by the Wizard minipreparation (Promega). The plasmids are used as templates in a polymerase chain reaction using the cloned primers and the amplified is analyzed by electrophoresis in agarose and ethidium bromide to stain. Plasmids carrying the appropriate size amplifications are sequenced (Abl 310 sequencer) to identify the clones, which contain the desired sequence. The clones are secured and used for subsequent production. A similar strategy can be used to clone any gene of interest.

It is particularly preferred that the protein be a vertebrate protein, for example, a human, a murine, a rat, porcine, bovine, or poultry protein. • In another embodiment, the invention relates to a process for producing a plurality of functional proteins where at least one of the proteins is from the immunoglobulin superfamily, and the plurality of proteins, when functional, contain at least one intramolecular or intermolecular disulfide bond, the process comprises the steps of 15 providing a bacterial cell comprising a plurality of genes encoded each with a protein, all genes being expressible in said cells, culturing the cell under conditions where the genes are expressed, (iii) isolate the proteins of the cell under conditions that do not change the bonds of -Jjri .Ajii »disulfide generated by the cell, and optionally, purify it, (iv) subjecting the proteins to isolation for the folding treatment.

In this embodiment, the protein can be a fusion protein or it can be two separate proteins, that is, proteins co¬ 10 expressed.

• A further embodiment relates to a process for producing a protein of the functional immunoglobulin superfamily, which has at least one disulfide bond when it is functional, the process comprises the steps of i) providing a cell comprising a gene for coding the protein, the gene is expressible in said cell, the protein being expressed as an aggregate, (ii) culturing the cell under conditions where the gene is expressed, ^ IMi Mtatf (iii) isolate the aggregated protein from the cell under conditions that do not change the disulfide bonds generated by the cell, and optionally, purify it, (iv) subjecting the isolated protein to the folding treatment.

A denatured protein can occur in many different conformations, not having a distinctive conformation, whereas a protein folded in an aqueous solution occurs in one or in some different conformations. One of the essential characteristics of this invention is that they avoid the conventional solvation of the aggregated protein effected by the (denaturation under reduced conditions that leads to a fully deployed protein.) The subsequent refolding, in order to generate a correctly folded protein, is complicated because to the requirements for the recreation of the correct disulfide bonds The method of the invention is different because it has surprisingly been found that the proteins present in the aggregates, for example, the , ,,. . ^ j? sa inclusion bodies that appear to be present in a functional way, have correct disulfide bonds, and the task in this way is to solubilize them without breaking the bonds of • 5 disulfide. The present inventors have found that the denatured solution of the protein should • be run under non-reduced conditions without altering the state of oxidation reduction of the protine. Using denatured proteins with 10 the correct disulfide bonds leads to a simplification of the refolding process which can now be as simple as a dilution of, for example, urea without adding a reduction coupling oxidation. The folding can, without However, to be assisted by other proteins such as chaperones; in the case of class I MHC, it can be assisted by ß2m and / or peptide. In addition, the folding treatment according to the invention can be carried out for certain proteins, 20 for example MHC, essentially in the absence of an oxidation reduction coupling such as GSSG / GSH.

The isolation can be performed by breaking the cell, separating the aggregates such as inclusion bodies (for example by centrifugation), optionally washing, extracting the aggregates (for example inclusion bodies) in a denaturing agent (for example, urea or guanidine-hydrochloride). , or by other methods known to a person skilled in the art) leading to the extraction of the soluble protein. This is a schematic summary of the isolation process of (iii; which can be modified or followed, for example, by a purification step that will be evid for the person skilled in the art. It is now particularly convenient, for currently preferred MHC molecules, to add a purification step since many associated non-covalent molecules including oligo-peptides can be removed. Such a purification may be ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction, precipitation, filtration, centifugation and other methods known to the person skilled in the art.

The folding begins by diluting the denaturing agent (for example urea) to the extent that it reaches the folding of the protein. Preferably, the folding step of the process of the invention (iv) is carried out in an aqueous medium which can comprise at least one buffer compound. The protein can then be subjected to a purification procedure as described above.

In the process of the invention, a cell comprising a gene encoding a heterologous or homologous protein, whose gene is expressible in said cell, can be any cell. Preferably, the cells are selected from the group consisting of bacterial cell, fungal cell, yeast cell, animal cell, and plant cell. More preferably, the cell is a cell of bacteria selected from the group consisting of gram positive bacteria and gram negative bacteria. In a presently preferred embodiment, the gram negative bacterium is E. coli including a strain BL21 or a derivative thereof or a strain XA90 or a derivative thereof.

It is contemplated that another useful cell may be a cell that is genetically modified to * ~ t * r * kfl, - -. - t t ..... AA ^ á »,», have a less reduced intracellular environment than an unmodified cell of the same strain, for example, the cell that has been modified to have a reduced activity or lacking a reductase • of thioredoxin or an enzyme that has a similar effect on the sulfhydryl reducing potential of the cytoplasm such as txB-mutant. Another useful strain may be a strain that is capable of biotinylating the protein, that is, that is capable of 10 Biotinylate a protein having a biotinizing sequence. The protein can be modified in • live or in vitro, for example, phosphorylating, glycose, acetylating, amidating, or modifying in any other appropriate way. The expressed protein can be localized intracellularly, per iplasmatically or ext race locally. • The insertion of the gene encoding the functional protein is carried out by any conversion technique for the introduction into a cell of nucleotide sequences, for example, by transformation, transfection, or transduction. He 25 gene can be inserted into the chromosome of the cell "^ *" M "* to host typically by means of transposome or by a recombination event, or can be introduced episomally by means of appropriate vectors.

^ B purposes include pET vectors, such as T7 promoters.

It will be appreciated that the gene can be produced in the host cell alone or in combination with 10 additional nucleotide sequences including sequences that regulate gene expression such as promoter sequences, promoter regulatory sequences, enhancer sequences, sequences encoding the repressor substance including 15 ARM ant i -sensible, or determination sequences. In order to increase the protein produced, multiple copies of the gene encoded for the functional protein can be introduced into the cell • Guest. It is also possible to enter sequences 20 that encode for the chaperone proteins or regulatory sequences of the expression or functionality of the native chaperone proteins, or sequences that encode the product of the gene that results in the glycosylation of the protein functional. The promoter can be constitutive or inducible.

The process according to the invention will be advantageous in one or more aspect of the protein product. It may be that the performance of the functional protein produced in accordance with the process in relation to the performance of the functional protein obtained under essentially similar conditions but where step (iii) is carried out under B conditions that do not change the disulfide bonds generated by the cell, will be increased by 10%, such as minus 20%, at least 40%, at least 50%, at least 70%, or at least 100%. Alternatively, it may be that the process velocity compared to when step (iii) is performed under conditions that do not change the disulfide bonds generated by the cell, be at least • 10% faster, such as at least 20%, at least 20 40%, at least 50%, at least 70%, or at least 100%. It is contemplated that the speed increase may in fact be at most 2 times, 5 times, 10 times, 100 times or 10000 incremented. In a presently preferred embodiment, the speed is increased by at least 50 times. • *! -t ™ tirw? ~ * - 'i-m •' i. , itt ^ jü ??? Finally, it may be that the purity of the functional protein produced in accordance with the process in relation to the purity of the resulting functional protein obtained under essentially similar conditions but where step (iii) runs under conditions that do not change in the disuifuro bonds generated by the cell, is increased by at least 10%, such as at least 20%, at least 40%, at least 50%, at least 70%, or at least 100%.

With reference to the examples, in particular the MHC A2 protein, the folding efficiency can be at least 40%, while the MHC Db protein has a folding efficiency that is considerably higher, that is at least 50%. The% is measured in the active protein immediately after folding by comparing the amount of input protein in question in the folding process. It is contemplated that when the process according to the invention has been optimized with respect to the protein in question, then at least 25% of the protein of the immunoglobulin superfamily produced is obtained in a functional form.

Preferably, the protein comprises non-disparate cysteine residues. However within the scope of the invention is an embodiment wherein the protein comprises a • 5 dispar cysteine residue. The protein may comprise at least 2, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, 10 at least 16, at least 17, at least 18, at least 19, at least 20 cysteine residues. Preferably the protein comprises even a number of cysteine residues. More preferably, the protein has a maximum of 20, such 15 maximum 14, maximum 10, maximum, maximum 5, maximum 4, maximum 3, maximum 2 cysteine residues. The protein is preferably capable of having at least 1, such as at least 2, at least 3, at least 4, at least 20 5, at least 6 disulfide bonds. More preferably, the protein is capable of a maximum of 20, such as maximum 15, maximum 10, maximum 8, maximum 5, maximum 4, maximum 3, maximum 2 disulfide bonds. In a preferred embodiment, the gene is a derivative of a naturally occurring gene. The derivative can be obtained by replacing at least one codon that is used more frequently by the host cell than in the one originally present where the codon codes for the same amino acid. Generally, the gene is under control of a DNA regulatory sequence not naturally associated with the gene. However, it can also be under the control of its promoter. In a presently preferred embodiment, the bacterium cell is transformed with an expression vector selected from the group consisting of pET vectors, eg, T7 promoters. Other vectors will be apparent to the person skilled in the art.

A more important aspect of the invention is a stable peptide free MHC protein which are obtained by a process according to the invention. A stable peptide-free MHC protein has not been previously generated by any of the methods within the art. Although it is claimed (Matsumura et al., 1992) that empty molecules can be obtained from TAP-deficient eukaryotic cells such as T2 or insect cells, the peptides are extracted and characterized from such preparations (Wei and Cresswell, 1992 Henderson et al, 1992 i, that is, those preparations that are not truly empty One of the uses of stable peptide free MHC protein is provided for a highly efficient production of pure homogeneous MHC peptide complexes. "Stable" means that the heavy chain in isolated form in urea can be stored for at least 3 months at -20 ° C at 50% glycerol.The half-life of the complex is currently greater than six months The stability of the functional MHC complex in aqueous solution, that is, the heavy chain and ß2m in 1: 1 is being investigated, however, it is known that the half-life in the heavy chain in the presence of an exce or ß2m It is stable in an aqueous solution for about 2 days at 4 ° C.

Another important aspect of the invention is a kit comprising an MHC class I heavy chain and a ß2m chain allowing the recipient to produce a functional class I MHC protein to which a peptide, which is capable of binding to said MHC class protein I, can be added by initiating the generation of a functional MHC protein and I protein. The MHC proteins will preferably be produced by the method of compliance of the invention. In one embodiment, the kit comprises reagents that will allow the end user to determine the binding of any peptide of their choice using detection systems such as enzyme linked immunosorbent assay (ELISA), radio immuno assay (RIA), and other known ones by the person skilled in the art. In another embodiment, the kit comprises the oligomerization of MHC proteins such as two, three, four or more. In a specific embodiment the kit comprises an additional reagent added by a label making the case appropriate for diagnostic purposes. The label is preferably selected from the group consisting of luorochromes, enzymes, chemoluminents, and radioactive labels.

A preferable use of the process of the invention is in the manufacture of MHC, in particular the peptide-free MHC molecules. A preferred use for stable peptide free MHC protein is • in the analysis of the effect of changing an amino acid in the MHC in the specific assembly of said MHC as assessed by an analysis using a peptide library close to being a synthetic or recombinant. Such a combination of 10 MHC point mutations followed by a specificity analysis by the peptide collections constitutes a novel approach to the examination of the structure-function relationship. Additional uses of the invention are described below. 15 The use of recombinant MHC I molecules Analysis / diagnosis (in particular by ELISA and / or FACS) 20 a.l) Quantification of the peptide-MHC interaction. It is of considerable interest to measure the interaction between the peptide and the MHC. Any putative T cell epitope should be verified by MHC binding, preferably in an assay ~ », *? * ÁG ?? iL ?. ^ »^. A». . A i. ... ...., «« ** flsü quantitative prti. The current methodology for measuring such interactions is hampered by poorly controlled assay systems, the lack of empty MHC molecules and / or the requirement for • 5 mark at least one of the components. Empty molecules are highly active and easily adaptable for highly controlled biochemical test systems of the RIA type (Buss et al., 1995). The empty molecules 10 are also adaptable for sub-detection by • an ELISA approximation. In a preferred embodiment, this ELISA assay involves the capture of the specific vessel anti-MHC antibody and the detection of the anti-MHC antibody. 15 of specific vessel allowing a highly sensitive, quantitative non-radioactive detection. Other ELISA assay designs (e.g. wraparound affinity tags) are known to those skilled in the art. 20 a.2) Enumeration of specific T cells for quantitative and qualitative characterization of T cell populations. Mark Davis et al. In 1996 (Altman et al., 1996) demonstrated 25 that the pure, recombinant class I MHC peptide complexes generate an agitated biotylation signal linked to the C terminus of the heavy chain. After the enzymatic biotylation process, these complexes can be damaged. • 5 with st reptavidin marked with phyrereitine. These labeled, multimeric MHC class I peptides will subsequently be used to label T cells in a peptide-specific MHC I-restricted manner. The stability of The T-cell receptor for the peptide complexes- MHC I is generally intended to be too low to effect a stable biochemical bond. However, after the ramerization, the avidity of multi-site complexes is sufficient to 15 perform a biochemical link. In this way such tetramers can be used to be counted by anal i. fluorescent activated cell (FACS) lottery of the number of T cells in any given cell suspension. This has proven Subsequently, the older methods for specific T-cell count, limiting their dilution analysis, are completely incorrect and underestimate the number of specific T cells. In this way, it becomes imperative 25 tant for from the scientific point of view, but also from a publication point of view, make a "tetramer" analysis. The Davis method has two major problems: its difficulty in producing large quantities of ^ m 5 pure MHC molecules, and that the biot initiation process is expensive, uncomfortable, and in particular, requires an extended incubation at 37 ° C. Many peptides are not associated with the MHC at the time of this last incubation, this may explain the result varied even within the same laboratory. The present patent application describes a method by which peptide-MHC I complexes can be generated in a fast and efficient process leading to the formation of a clean minimal back complex. It is envisaged that the MHC I molecules produced according to the present method can be transported and stored in a way that allows the generation of a commercial kit, which also allows the production of the final peptide-MHC I complex to be given in any laboratory not expert using any choice of the end user of the relevant peptide. In contrast, the previous ways of producing MHC I required considerable knowledge and experience of the protein in the «« ?? jtkM.AMH &léiik k? A .... . .. -. -, - .. . " . _. , ^ AA «t > ^ ...- Aa molecular biology. Finally, many better forms of MHC labeling than the enzymatic biotylation process can be observed (Gallimore and collaborators, 1998, Walter and cola oradores, 1998). a.3) Enumeration of specific T cells enabled for immune manipulations to be adequately monitored. Any immune manipulation (for example, vaccination) is necessary for acute and specific monitoring. The technology of "tet amero" previous is up to date in the golden standard of the specific evaluation, clinical and commercial of immune manipulation. b) Scientifically bl) Functional and structural determination of the specificity of MHC I molecules. MHC molecules are central players in the generation of responses mediated by all T cells. Considerable efforts have been directed to the understanding of the function and specificity of MHC molecules I For all these purposes, access to functionally active MHC I molecules is necessary. The production of MHC I molecules from natural sources have several serious disadvantages (discomfort, expensive production, and even yields in small amounts of • 5 impure MHC). The method describes a way for an easy, fast and highly efficient production of peptide-MHC I complexes. The previous methods, which are used to produce complex peptides-MHC I include a step where an excess of peptide is 10 offers the MHC I under refolding (Garboczi et al., 1992). In this way, the resulting complexes are previously occupied with a peptide and therefore are not readily available for the n ovo peptide linkage. From In accordance with the method described, the MHC II molecules produced can be used immediately to bind the peptide, and therefore are useful for any analysis, including specificity analysis, peptide bonding. Combining the 20 recombinant MHC I molecules with the approach to the collection of recently published peptides (Stryhn et al., 1996), the specificity of any MHC I molecule including mutated MHC I molecules can be examined in detail. 25 It should be emphasized that such detailed analysis it also leads to the improvement of peptide bond predictions (Stryhn et al., 1996). b.2) Functional and structural determination of • 5 the specificity of T cells. Peptide-MHC I complexes can be generated rapidly, purely and efficiently by the method described. Such complexes can be used to analyze the structure of the T cell receptor in the 10 interaction of said peptide-MHC I complex, and using peptide variants and / or MHC I variants it will also be possible to execute a functional analysis of the specificity of the T-cell receptor. 15 b.3) Specificity of T cell manipulation (induction or blocking). The peptide-MHC I complexes described herein will be able to interact with the T cell receptor in a 20 given target cell. In order to stimulate the cell, its T-cell receptors must cross-link. In this way, it can be expected that the polymerized MHC I complexes (said "tet ameros") can appropriately stimulate the 25 peptide specificity, the T cells restricted from ^^ i * n * ¿? Iskx? * t. *. '. u > > MHC I, while the peptide-MHC I complexes can block the same cells. b.4) Monitoring of the specific effect of • 5 immune manipulations (for example, vaccination). Any research so far to manipulate the immune system of the T cell will be, except for further improvements in technology, dependent on the existence of the "tetramer" technology (or 10 similar to the tetramer). The ability to induce B specific predetermined responses can be sharpened and easily determined, and by modern FACS analysis even if additional analyzes in whose sub-populations are affected 15 too much The tetramer technology will therefore be essential for future developments in immune mani ulation.

Wt b.5) Purifications of specific T cells. 20 Tetramers will allow the efficient purification of MHC I restricted T cells from specific peptides. As an example, if the tetramers through their biotin are coupled to paramagnetic beds, the total 25 purification of the corresponding specific T cells using a magnet. The specific T cells can then be completely eluted and used for analysis or expanded and used for additional immune manipulations (eg, • 5 t ras Fibers of adoptive T cells). This constitutes a vast improvement compared to the present cloning technology which is extremely annoying and slow. 10 c) Therapy c.l) Development of vaccine. The effects of vaccine development can be crudely subdivided into a direct or indirect effect. A direct effect of the described technology will be the therapeutic application of the principle mentioned in b3, where a highly specific and highly efficient activation of the specific T cell (and subsequently of the immune system) is seen on the basis of the MHC I molecules carried of peptide, isolated administered in a stimulating manner (reticulated as "tetramer", or beds etc.). One effect or indirect is caused by the improved identification of candidates for vaccines that will be the result of the technology described. Enabling g ^ g ^ i ^^ jj ^^^^^ g ^ g ^^^ -, ^^^^^ '^ «^ g ^ - ^^ iM > Based on a large-scale analysis of all human MHC I molecules, predictions of peptides derived from pathogens capable of MHC binding can be improved, and allowing easy validation of such predictions (MHC-specific epitope analysis). c.2) Treatment of autoimmune diseases. The effects in the treatment of autoimmune diseases can be crudely subdivided into a direct and indirect effect. A direct effect of the technology described will be the therapeutic application of the principle mentioned in b3. where a highly specific blockade of specific T cells (and subsequently of significant parts of the immune system) is seen based on peptide-loaded MHC I molecules, isolated, administered in a non-stimulatory manner (ie, as non-cross-linked soluble complexes). An indirect effect will be caused by the improved identification of autoimmune diseases induced by cand data that will result from the described technology. By enabling detailed, large-scale analyzes of all human MHC I molecules, the predictions of the peptides will be improved . , L. . .. ".,. ,. . . ^ j ^ tüA derivatives of these proteins capable of binding to the MHC, and allowing easy validations of such predictions. c.3) Purification of T cells by adoptive transfer. As detailed in b5, T cells can be specifically shipped using "tetramers," and thus also be raffled off (ie, by magnetic beds) leading to the preparation of pure T cell populations of predetermined specificity. Such populations of T cells can then be expanded and used, for example, for infections against adoptive transfers or oncogenic diseases. c.4) Treatment of cancer. Many cancers are associated with mutated tumor oncogene / suppressor genes, or with deregulatory / suppressor oncogenic genes. The resulting change in cellular metabolism can be detected by the immune system. The following examples of deregulations lead to an altered level of a completely normal protein itself. Still the altered level of the same protein that becomes immunogenic with rejection of the tumor has been demonstrated ^^^^^ _ l ^ Ma ^ * _ áaé_ in such cases (Vierboom et al., 1997).

Mutations and deregulations can be detected as exemplified, but not limited to, by genetic analysis (eg, "single stranded" conformational polymorphism, "selective polymerase chain reaction" or "differential display") or protein analysis (mass spectrometry carried out by proteome analysis). Any protein identified here will undergo the MHC-specific epitope analysis described above and a specific cancer treatment can be attempted as detailed in cl and c3.

LEGEND OF THE FIGURES Figure 1 : Production of HLA-A * 0201. Recombinant XA90 cells encoding the A2 (1-275) trickle induced with 0.4 mM IPTG. The production was analyzed in 15% SDS-PAGE under reduced conditions.

Track 1: the markers as indicated. Route 2: cellular proteins before induction. Route 3: cellular proteins 3 hours after induction. Lane 4: HLA-A * 0201 after purification using anion exchange chromatography.

Figure 2: 10 The fractions of anion exchange with • HLA-A * 0201 recombinant heavy chains of solubilized urea inclusion body proteins were analyzed on a 15% SDS-PAGE under non-reduced conditions. The corresponding fractions for purifying the peptide linking monomers were analyzed (fraction numbers indicated above, compared to Figure 4). This shows that recombinant HLA-A * 0201 migrates as two distinct proteins 20 around 31-32 kD; the protein band 2a and 3, both with intact disulfide bonds.

Figure 3: - ~ l * & ~ ?? k u. . • i. .. «. .. ^ - M SDS-page analysis of recombinant HLA-A * 0201 molecules purified in the presence or absence of DTT. Lane 1: HLA-A * 0201 heavy chain highly purified and functional results in • Starting from one fold per dilution process (not reduced). Lane 2 and 3: the heavy chain of HLA-A * 0201 anion exchange in non-reduced condition (lanes 2a and 3). In track 3 it is shown that both protein bands are partly 10 reduced in band 1 by the reductant present in path 4. Via 4: the heavy chain of HLA-A * 0201 of anion exchange in reduced condition revealed the band of protein 1, which migrates slower than the protein band 2a and 3. 15 Figure 4 The anion exchange fractions analized by the amount of proteins eluted and 20 that correspond to the binding capacity of the peptide. The concentrations were determined by BCA and measured in OD562 (full line). The same fractions were also tested for the peptide linkage. Around 30% of the amount 25 total protein applied to the unbound column (basic and neutral charged proteins). These proteins correspond to the bacterial ancestor proteins co-purified with the inclusion bodies. Recombinant HLA-A * 0201 molecules were eluted with about 200 mM NaCl and identified by peptide linkage analysis (dotted line) and SDS-PAGE analysis (Figure 3).

Figure 5: The immunoprecipitation of the folded recombinant HLA-A * 0201 molecules using specific antibodies.

The heavy chain complexes of recombinant HLA-A * 0201, b2m and radiolabelled peptide were incubated with monoclonal antibodies either against molecules of HLA-A * 0201, H-2Kb or H2Db at 4 ° C. Subsequently, protein A was added to the precipitated immune complexes. The precipitate was repeatedly washed and counted for its radioactivity. Only specific antibodies HLA-A * 02C1, BB7.2 and W6 / 32 can- precipitate the labeled radical peptide bound to the recombinant HLA-A * 0201. This was an interaction that can not be measured between recombinant HLA-A * 0201 and antibodies with irrelevant specificity.

Figure 6: 51 peptide linkage for the heavy chain of recombinant HLA-A * 0201.

Increased doses of recombinant HLA-A * 0201 heavy chains were incubated with b2m (lμM) and a radiolabelled peptide (2 nM) trace amount for 4 hours at 18 ° C. The degree of complex formation was determined by spun-column chromatography G25.

Figure 7: The affinity of peptide interaction with recombinant HLA-A * 0201 5 nM of recombinant HLA-A * 0201 was incubated with radiolabelled peptide and b2m trace amounts (1 μM) and increased concentrations of unlabeled peptides with specificity to HLA-A * 0201 and H2-Kk, respectively. The reactions were incubated for 4 hours at 18 ° C, and the degree of complex formation was determined by spun-column chromatography G25.

The concentration of ligand bound against the proportion of free and bound ligand was plotted to obtain a Scatchard trace (inserted).

Figure 8: The b2m-dependent peptide linked to recombinant HLA-A * 0201.

The heavy chains of recombinant HLA-A * 0201 were incubated with an indicator amount of peptide and increased concentrations of human and mouse b2m as indicated. The reactions were incubated for 4 hours at 18 ° C, and the degree of complex formation was determined by spun-column chromatography G25.

Figure 9: Increased doses of recombinant HLA-A * 0201 heavy chains were incubated with human radiolabeled b2m in the presence or absence of specific peptides (10 μ) for 4 hours at 18 ° C. The reactions were incubated for 4 hours at 18 ° C, and the degree of complex formation was determined by G50 spun column chromatography.

Figure 10 The dissociation of b2m from the HLA-A * 0201 reconbinant complex. Purified spiked complexes of radiolabeled heavy chain b2m with or without peptide were mixed with 3 μM unlabelled b2m and incubated for the indicated time at 4 ° C. The degree of dissociation was determined by Sephadex G50 spun column chromatography.

Figure 11: Tetramer stain evaluated by analysis FACS. The cells analyzed are CD8 positive T cells from either mice carrying a transgene for a specific T cell receptor by the KAVYN-FATM peptide in association with the H-2Db or non-transgene control H-2Db mice. The analysis was performed with phycoerythrin-streptavidin that generates tetramers involved with either the relevant KAVYNFATM peptide 5 in a complex with recombinant H-2Db and biotinylated b2m, or with the irrelevant peptide FAPGNYPAL in a complex with H2Db and biotinylated b2m. The FACS analysis was performed with the tetramer stain on the x axis and the CD8 stain on the 10 the y axis. The amount of positive tetramer, • CD8 positive cells observed in the upper right frame. The percentage of total cells that are both tetrameric and CD8 positive is calculated to the right of the column and is given 15 directly in the upper right box.

Complex T Cells% of tetramer + and CD8 + • Figure HA Relevant Relevant 53% Figure 11B Relevant Irrelevant 2% Figure 11C Irrelevant Relevant 1% Figure 11 D Irrelevant Irrelevant 3% EXAMPLES Materials and methods • Urea, phenylmethylsulfonyl fluoride (PMSF), isopropyl-b-D thiogalactidase (IPTG), bicinconic acid solution (BCA) and tr i s [hydroxymethyl] aminomet anus (tris) was purchased from Sigma. The Sephanex G50 and the material of 10 Q-Sepharose fast-flow anion exchange was purchased from Pharmacia, Sweden.

Production of MHC murine and human class I heavy chains. The heavy chain of recombinant HLA-A * 0201 (1-275) in XA90 cells was of the type donated by Drs. Wiley and Garboczi. The XA90 cells of • a culture overnight were inoculated and 20 grew at 37 ° C for production in 200 ml of Lur ia-Bert ani medium in 100 μl / ml of ampicillin.

The recombinant H-2D (1-276) in the pGMT7 vector (a pET derivative) was of the type donated by the 25 Doctor Gallimore. The H-2Db containing plasmid was transformed into Escherichia coli strain BL21 (DE3) (Novagen) and grown at 37 ° C in 200 ml of Luria-Bertani medium containing 100 μl / ml of ampicillin. The cells of a nocturnal culture are • 5 inoculated and grew at 37 ° C in 200 ml of Luria-Bertani medium in 100 μl / ml of ampicillin for production.

The expression protein was induced in phase 10 of log medium (A600 = 0.6) with 0.4 mM isopropyl-b- • d-thiogalactosidase (IPTG). The cells were harvested by centrifugation after 3 hours. The cells were suspended in 10 ml, 20 mM tris buffer, pH 8.0 and 1 mM EDTA, and stored at -20 ° C.

Isolation of inclusion bodies and purification of heavy chains of HLA-A2 * 01 class I • recombinant. Preparations of frozen cells (from 200 ml cultures) were sonicated in 10 ml of 20 mM tris, pH of 8 with lysozoma (100 μg / ml), EDTA (1 mM), PMSF (50 μg / ml) and HE 25 incubated 20 minutes at room temperature.

Subsequently, DNAse (10 μg / ml) and MgCl (10 mM) were added. After clearing, the inclusion bodies were partially purified by centrifugation in 15 minutes at 10,000g. The • 5 pellets containing the inclusion bodies were washed 3 times in the tris buffer, the pellets are finally solubilized by a 2 hour incubation in 3 ml 8M urea, 20 mM tris, pH 8.0 with PMSF and EDTA a 4 ° C. He Insoluble material was removed by centrifugation.

• The supernatant was harvested and passed over 0.22 um filters before storing at -80 ° C. These preparations contain heavy chains of HLA-A * 0201 class I with a purity of about 15 60 - 80% estimated SDS-PAGE. The heavy chains of HLA-A * 0201 class I were purified using an anion exchange column (1 x 25 cm) (fast flow, Pharmacia). The • preparations with partially heavy chains Purified samples were diluted 5 times with 8 M urea, 20 mM tris, pH 8.0 and the anion exchange matrix was applied. The column was washed with 20 ml 8M urea, 20 mM tris, pH 8.0 and the proteins were eluted in a gradient from 0 to 500 mM NaCl 25 in 8 M of urea, 20 mM of tris, pH of 8.0 of the buffer solution. Eluted proteins were monitored by determination of BCA protein, SDS-PAGE analysis and peptide binding capacity. Fractions that contain proteins from • 5 highly purified monomer heavy chain and high peptide binding capacity were pooled and stored at -80 ° C.

III. ? Purification of b2m antibodies and 10 monoclonal.

• The human and recombinant mouse b2m was produced and purified as described previously (Pedersen et al., 1995). 15 Monoclonal antibodies, W6 / 32 (a-HLA class 1), BB7.2 (a-HLA-HLA-A * 0201), 11-4.1 (a-Kk), 28- 14-8S (aH- 2Db), B22-249 (aH-2D), were produced as ascites and purified by protein A chromatography (most of these hybridomas are 20 ATCC).

Radio-iodination of b2m and peptide HLA-A * 0201 and H-2Db of specific peptides were purified by reverse phase HPLC chromatography and lyophilized. The peptide (1-2 μg) was radiolabelled for a specific activity of 60 mCi / μg as previously described (Olsen and Colaidors, 1994). The peptide fraction • 5 linkable to MHC class 1 recombinant or narive is routinely 80%.

Electrophoresis 10 One-step SDS- • polyacrylamine gel electrophoresis (PAGE) was run on homogeneous polyacrylamide gels (15% I. Samples were baked in Laemmli sample equalizing solution with or without 50 mM. 15 of DIT before the SDS-PAGE analysis. The proteins were stained with Coomassie Blue R-250.

Peptide Binding to Recombinant Class 1 HLA-A * 0201 Molecules (Indication Link) 20 The denatured recombinant HLA-A * 0201 heavy chains of the anion exchange purification were tested for their peptide binding ability in the presence of b2m .

The peptide bond for recombinant heavy chains is conducted essentially as a conventional folding by the dilution test (Garboczi et al., 1992) except that the amount of radiolabelled peptide is an amount • 5 indicator, that is, around 2 mM. Typically, 1 microliter of denatured heavy chain solution of anion exchange purification fractions is diluted 100-fold in a folded buffer solution 10 described below. The reaction was incubated for at least 4 hours. The peptide bond was examined by spun-column chromatography G25 Sephadex (Buus et al., 1995). All experiments have to be conducted two or more times. The 15 optimization of the peptide linkage for HLA-A * 0201 and recombinant H-2Db, revealed a folded buffer solution consisting of 20 mM Tris pH of 7. 150 mM NaCl, 1 mM EDTA, 50 μg / ml PMSF , 1 μM of b2m and 1-2 nM of indicator 20 péptrdo. It will be noted that classical folding agents such as L-arginine and bacterial chaperonins such as GRO-EL / ES, have a negative impact on the peptide bond (data not shown). The normal ranges of 25 GSH / GSSG concentrations (e.g., 1.8 mM / 0.2 mM) have no effect on peptide interaction. Peptide bond analysis in a temperature range from 4 ° C to 37 ° C indicates an optimal bond at 18 ° C. Link analysis • Peptide in a pH range of 5.5 to 9, indicates an optimal bond at a pH of 6.5 - 7.5. Kinetic studies show that the balance of the indicator peptide bond is established after 4 hours of incubation at 18 ° C. 10 Generation of functional class I HLA-A * 0201 molecules (folding to scale).

Heavy chain preparations of purified and denatured HLA-15 A * 02D1 (300-600 μg / mL) were folded by a 100 fold dilution in a folded buffer (seen above) to which the peptide is added to • a concentration of around 10 μM. The The reaction was concentrated 10-20 times at 4 ° C using an Amicon filter with a cut of around 10 kD. The concentrate was incubated at 4 ° C for 1 hour and cured for 15 minutes at 15,000 g. The supernatants were additionally concentrated 25 using Centricon units with a cut of dt küIllWMÉ &ttMEttl around 3 kD at 4 ° C. Centrifugation was repeated and the supernatants were applied to size exclusion chromatography (Sephadex G50) to exclude free b2m and peptide. Fractions containing functional HLA-A * 0201 class I were pooled and concentrated to a final concentration of about 1 mg / ml.

Results Expression and purification of the denatured recombinant HLA-A * 0201 heavy chain.

Recombinant H-2Db and HLA-A * 0201 were partially purified and partially purified as described by Garboczi et al. (Garboczi et al., 1992). Cells are induced with 0.4 mM IPTG at a cell density of about 0.6 and incubated for 3 hours at 37 ° C. Electrophoretic mobility of cooking and reduced samples with and without IPTG were analyzed on 15% SDS-PAGE gels. Yields of recombinant HLA-A * 0201 were estimated to be around 40-50 mg / ml correspond to the predominant protein band of around 33 kD in Figure 1, track 3.

To isolate the inclusion bodies, the 5 cells penetrate by sonication. The cells expressing recombinant heavy chains (from 200 ml of culture) were centrifuged and the pelleted ones were again solubilized in buffer solutions containing 20 mM of 10 tris, pH of 8, lysozyme, PMSF and EDTA.

• Subsequently, the DNA and MgCl were added. After clearing the solution (20-30 minutes at 22 ° C) it was centrifuged to pellet the inclusion bodies. The pellet was washed 3 times in 15 buffer solution tris pH of 8 and finally it was again solubilized in 8 M of urea and stored at -80 ° C.

• The partially purified proteins of the inclusion bodies were further fractionated using anion ion exchange chromatography. The reasons for this purification step is to enrich the preparation by recombinant HLA-A * 0201 molecules of high efficiency of folding and to avoid , k? 2É & G &g & ^^ ¿i ^^ ~ heterogeneity (minor contaminants, minor enzi.tiáticas chains of the heavy chains in the bacterium etc). • 5 The recombinant HLA-A * 0201 and H-2D monomers were eluted in a gradient (0-500 mM) of NaCl. The heavy chain monomers of and recombinant H2Db were eluted at about 200 mM and 350 mM NaCl, 10 respectively. Purity, concentration and functionality of the purified heavy chains were analyzed in SDS-PAGE (figure 1, lane 4 and figure 2), and by assays for BCA protein determination and by peptide bond analysis 15 indicator (figure 4).

Anion exchange purification and non-reducing SDS-PAGE analysis showed that most, if not all, of the HLA-A * 0201 protein The recombinant, solubilized from the isolated inclusion bodies, was oxidized, ie containing di-sulfide bridges. As shown in Figure 2, the heavy chains of recombinant HLA-A * 0201 migrated as two distinct bands 25 (hereinafter called 2a and 3, respectively) in a non-reduced SDS-PAGE analysis. Protein 2a will not be separated from protein 3 using hydrophobic or anion exchange, size exclusion chromatography. Under reduced conditions, the two heavy chains of HLA-A * 0201 co-migrated slowly as a band called single protein band 1 (Figure 3, compare via 2 and 3 with pathway 4). This band corresponds to the dominant HLA-A * 0201 protein band in Figure 1 (lane 3). In conclusion, the proteins are packaged in the inclusion bodies with the intact di-sulfide bridges charged in at least two different monomers. Folding experiments, as described below, clearly show that only protein band 2a will be folded into a functional recombinant HLA-A * 0201 complex (FIG. 3, compare via 1 and 2). The protein band 3 is unfolded and added during folding by the dilution process. Presumably, the protein band 2a containing di-sulfide bridge (s) is correct, while the protein band 3 is incorrect. Also, the chemical cross-linkage of the radiolabelled peptide for the heavy chain of HLA-A * 0201 recombinant identifies the band of protein 2a as the receptor peptide (data not shown).

The rapid migration of proteins packaged in • 5 bodies of inclusion seems to be a general phenomenon. A rapid migration of widely different proteins has been observed as human b2m, the domains of the gamma and epsilon chains of the CD3 complex proteins, H2-Kk chains, MHC 10 class II alpha and beta in a non-reducing SDS-PAGE analysis. Also, recombinant H-2Db molecules migrate at high speed under non-reducing conditions (data not shown). In comparison with heavy chains 15 of HLA-A * 0201, heavy chains of unreduced H-2Db migrated as a single fast migrating band which migrates slowly in reducing SDS-PAGE analysis (data not shown). Linkage of radiolabelled peptides to denatured partially purified denatured recombinant HLA-A * 0201 heavy chains. k »! iaM ~ ~ k ~, 1.

The recombinant heavy chains fractionated by anion exchange chromatography were tested for their ability to bind specific radiolabeled peptides aggregated during a folding by the dilution process (see materials and methods). As shown (figure 4) there is a good correlation between appearance of the HLA-A * 0201 reconbinant heavy chain mononer (bands 2a and 3) and the ability of the 10 peptide link. The purified heavy chains of the urea preparation with the reducing agent (more than 0.1 mM of DDT) do not bind to the peptides after the biochemical purification even in the presence of GSH / GSSG. In this way, the The ability of the peptide bond is related to heavy chains with preformed di-sulfide bridges.

• Both the heavy chains of recombinant H-2D 20 and recombinant HLA-A * 0201 bind to the specific radioligand which can be inhibited by specific peptides, but not by unbound peptides. Recombinant HLA-A * 0201 in a complex with radiolabelled peptides may be precipitated with specific antibodies against j ^ 2 ^^^ gS?! s5 £ *** ^^^ á & ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ but not with antibodies against H2-Kk and H-2Db molecules the correct HLA-A * 0201 (figure 5).

Linkage of radiolabelled peptides for highly purified denatured recombinant HLA-A * 0201 heavy chains.

The MHC class I functional generation of a denatured state of heavy chains with pre-formed di-sulfide bridges was evaluated using a radiolabeled peptide and radiolabelled b2m.

The fully purified HLA-A * 0201 heavy chains corresponding to the protein band 2a are obtained by denaturing functional recombinant HLA-A * 0201 using 8 M urea. The denatured proteins were fractionated by size exclusion chromatography (Sephadex G50) in a tris buffer solution of 20 mM, pH 8, with 8 M urea and the heavy chain was harvested in the void volume.

The binding of radiolabelled ligands (peptide or b2m) was completed by dilution in solution .. * double shock absorber as described above. A dose of response (Figure 6) shows a sensitive and highly efficient linkage of the radiolabeled peptide to the heavy chain • denatured during the folding process. In comparison, class I MHC molecules of conventional affinity require 10 to 50 high concentration folds to bind similar amounts of peptide. 10 The high efficiency of the peptide linkage was analyzed in an inhibition assay and by Scatchard analysis. As shown in Figure 7, recombinant HLA-A * 0201 only interacts with 15 specific peptides. The Scatchard analysis (Figure 7, inserted) reveals a simple straight Scatchard square and a constant equilibrium affinity of about 30 nM. Importantly, the active receptor fraction was 20 about 80-90% of the input protein. In this way, the heavy chain of HLA-A * 0201 is fully activated without concern for bacterial peptides or derivatives of any of the subsequent handling steps. In 25 comparison, Class I MHC preparations ?? i nMMJhiiiat. purified from conventional affinity were concerned with a peptide having a limited number of active receptors, typically 2%. • 5 The peptide bond is very dependent on b2m. As shown in Figure 8, increasing the doses of b2m added to the binding reaction facilitates the binding of the radiolabelled peptide. The absence of b2m completely prevents the link 10 peptide to the heavy chain. Also, the inverse reaction, that is, the b2m bound to the heavy chain shows the same dependence in the presence of specific peptides (Figure 9). The peptide n'o is however an absolute requirement as a 15 b2m that does not link; although with a low affinity, to the heavy chain in the absence of the peptide.

The effect of the peptides on the interaction of b2m is further analyzed in studies of 20 kinetics. The dissociation ratio of HLA-A * 0201 complexes consisting of radiolabeled b2m and heavy chains in the presence or absence of specific peptides is analyzed. The hetero-dimer complexes, that is, generated and maintained in In the absence of specific peptides, they dissociate rapidly with a half-life of about 4 hours at 37 ° C. The trimeric peptide-b2m heavy chain complexes, that is, generated and maintained in the presence of peptides, are stable comparison with a half-life around 14 hours. In this way, the peptides stabilize bound b2m. It is concluded that a HLA-A * 0201 compile is generated through a primary interaction between the heavy chain 10 denatured and b2m. the interaction generates a het e o-dimer, which expresses a high affinity peptide binding site. The peptides again increase the affinity of the linked b2m molecules resulting in complexes HLA-A * 0201 15 stable functional.

Generation of functional HLA-A * 0201.

Fractions with high peptide bonding capacity (about 15 ml corresponding to 60-70% of the mon era heavy chain fractions) were cultured and pooled for generation (folding) of functional HLA-A * 0201 molecules. The HLA-A * 0201 recombinant heavy chains were diluted in solution folded buffer as described above, 500 μg, in 1 ml, the partially purified heavy chain (corresponding to about 13 ml of bacterial culture) was added 5 to 99 ml of buffer and immediately concentrated using Amicon filters with a cut of around 10 kD. The concentrate of 5-10 ml (obtained within 1 hour) was incubated for 1 hour at 4 ° C before centrifugation at 15,000 g for 10 remove the aggregates. The supernatant was further cultured and concentrated to about 150-200 μl using centricon units with a cut of about 3 kD. After further centrifugation, the folded HLA-A * 0201 was purified 15 using size exclusion chromatography (G50) which retains the free b2m and peptides. Fractions containing assembled HLA-A * 0201 were pooled and concentrated using centricon to a final concentration of about 1. 20 mg / ml. The folding efficiency is around 40-50% calculated from the amount of denatured protein added. The yield of functional HLA-A * 0201, through the entire process, corresponds to about 10 mg of functional HLA- -25 A * 0201 / L of the bacterial culture. He The complete process of folding and purification can be conducted within 24 hours. It will be noted that the folding was without the addition of conventional agents such as L-arginine and GSH / GSSG. The former inhibits the folding of denatured HLA-A * 0201 heavy chains with preformed disulfide bridges. The latter does not effect the result. Folded recombinant HLA-A * 0201 molecules have been routinely tested on SDS-PAGE (Figure 3) and for their reactivity with specific antibodies (Figure 5). The recombinant H-2Db complexes generated by this process using binylated b2m are recently additionally assembled into oligomeric complexes using streptavidin. The H-2Db complexes ("tetramers") or oligomerics were used for FACS staining of specific T cells (Figure 11) and for staining of T cells as assessed by confocal microscopy. The latter demonstrates the specific link and the ability to intern.

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• Dominique Charron, EDK Press, 1997). 20 It should be noted that with regard to this date, the best method known to the applicant for carrying out said invention is that which is clear from the present description of the invention. 25 the invention.

Claims (1)

1. Claims Having described the invention as above, the content of the following claims is claimed as property. 1. A process for producing a protein of the functional immunoglobulin superfamily, characterized in that it has at least one functional disulfide bond, the process comprises the steps of (i) providing a bacterial cell comprising a gene encoding the protein, the gene is expressible in the cell, (ü) culturing the cell under conditions where the gene is expressed, 111 isolating the protein from the cell without reducing it, and, (iv) subjecting the isolated protein to a folding treatment. 2. The process for producing a plurality of functional proteins, characterized in that they include at least one of the immunoglobulin superfamily and where the plurality of functional proteins when functional has at least one intermolecular and / or intermolecular disulfide bond, the process comprises the steps of (i) providing a bacterial cell that • 5 comprises a plurality of genes encoding each of the proteins, all genes being expressible in the cell, (ii) culturing the cell under conditions where the genes are expressed, (iii) isolating the proteins from the cell without reducing them , (iv) subjecting the isolated proteins to a folding treatment. 3. The process for producing a protein of the functional immunoglobulin superfamily, characterized in that at least one disulfide is linked when functional, the process comprising the steps of (i) providing a cell comprising a gene encoded by the protein, the gene is expressible in the cell, the protein is expressed as an aggregate, (ii) culturing the cell under conditions where the gene is expressed, (iii) isolating the aggregated protein from the cell without reducing it. (iv) subjecting the isolated protein to a folding treatment. 4. The process according to any of claims 1-3, characterized in that the yield of the functional protein produced in accordance with the process in relation to the performance of the functional protein obtained under essentially similar conditions but where step (iii) is performed under reduced conditions, it is increased by at least 10%, such as at least 20%, at least 40%, at least 50%, at least 70%, or at least 100%. 5. The process according to any of claims 1-4, characterized in that step (iii) is performed under non-reduced conditions, the process velocity compared to when step (iii) is carried out under reduced conditions is at least 10. % faster. 6. The process according to any of claims 1-5, characterized in that the purity of the functional protein produced in accordance with the process in relation to the purity of the resulting functional protein obtained under essentially similar conditions but where the step (iii ) is performed under • 5 reduced conditions, it is increased by at least 10%. 7. The process according to any of claims 1-6, characterized in that the protein is a protein 10 of him immunoglobulin superfamily selected from the group consisting of antibodies, immunoglobulin variable (V) regions, immunoglobulin constant (C) regions, inmur oglobulin light chains, heavy chains of 15 Immunoglobulin, CD1, CD2, CD3, Class I and Class II histocompatibility molecules, ß2microglobulin (ß2m), lymphocyte-associated antigen-3 (LFA-3) and Fc? RIII, CD7, CD8, • Thy-1 and Tp44 (CD28), T cell receptor, CD4, Poly-immunoglobulin receptor, neuronal cell adhesion molecule (NCAM), myelin-associated glycoprotein (MAG), myelin P protein, carcinoembryonic antigen (CEA), platelet-derived growth factor receptor 25 (PDGFR), a factor 1 receptor that stimulates - ?? go MüiHUlÉ'iri? idiil? colony, aß-glycoprotein, ICAM (intercellular adhesion molecule), platelet and interleukins. 3. The process according to any of claims 1-7, characterized in that the protein of the immunoglobulin superfamily is a vertebrate, for example, a protein such as a human, a murine, a rat, a porcine, a bovine, or a bird protein 9. The process according to any of claims 1-8, characterized in that the protein of the immunoglobulin superfamily is an MHC. 10. The process according to claim 9, characterized in that the protein MHC is a human MHC. 11. The process according to claim 9 or 10, characterized in that the MHC protein is a MHC class I protein selected from the group consisting of a heavy chain, a heavy chain combined with a ß2m.- and a protein Functional mature Class I MHC; or a class II MHC protein selected from the group consisting of an α / β dimer and an α / β dimer with a peptide. 12. The process according to any of claims 9-11, characterized in that the MHC protein produced is obtained as a peptide-free MHC protein. • The process according to any of claims 7-12, characterized in that the folding process is terminated if at least 25% of the protein of the immunoglobulin superfamily produced is 10 obtains in a functional form. The process according to any of claims 1-13, characterized in that the protein in step (ii) is produced as inclusion bodies. 15. The process according to any of claims 1-14, characterized in that step (iii) is carried out under non-reduced conditions without altering the state of oxidation reduction. 16. The process according to any of claims 1-15, characterized in that the process further comprises a step wherein the protein isolated from step (iii) is subjected to a purification step before 25 of step (iv). ^^ ¡^^^^^^^^^^ 17. The process according to any of claims 1-16, characterized in that the folding treatment (iv) is carried out in an aqueous medium and at least one buffer compound. 18. The process according to any of claims 1-17, characterized in that the folding treatment is performed essentially in the absence of reducing agents, such as DTT. 19. The process according to any of claims 1-18, characterized in that the expressed protein is located intracellularly. 20. The process according to any of claims 1-19, characterized in that the expressed protein is located per iplasmically. 21. The process according to any of claims 1-20, characterized in that the expressed protein is trans-localized extracellularly. 22. The process according to any of claims 1-21, characterized in that the protein is expressed in a glycosylated form. 23. The process according to any of claims 1-22, • characterized in that the protein is expressed in a phosphorylated form. 24. The process according to any of claims 1-23, characterized in that the protein is glycosylated or 10 phosphorylated i n vi t ro. • 25. The process according to any of claims 1-24, characterized in that the protein does not comprise uneven cysteine residues. 26. The process according to any of claims 1-25, characterized in that the protein comprises 1 uneven cysteine residue. • 27. The process of compliance with 20 any of claims 1-26, characterized in that the protein comprises at least 2, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 25 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 cysteine residues. 28. The process according to any of claims 1-27, • characterized in that the protein still comprises a number of Cysteine residues. 29. The process according to any of claims 1-28, characterized in that the protein has as 10 maximum 20, such as maximum 14, maximum 10, • a maximum of 8, a maximum of 5, a maximum of 4, a maximum of 3, or a maximum of 2 cysteine residues. 30. The process according to any of claims 1-29, Characterized in that the protein is capable of having at least 1, such as at least 2, at least 3, at least 4, at least 5, or at least 6 disu fure bonds. 31. The process according to any of claims 1-30, characterized in that the protein is capable of having a maximum of 20, such as 15, maximum 10, maximum 8, maximum 5, maximum 4, at least 3, or at least 2 disulfide bonds. 32. The process according to any of claims 3-31, characterized in that the cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell and a plant cell. 33. The process according to any of claims 1-32, characterized in that the cell is a bacterial cell selected from the group consisting of a gram-positive bacterium and a gram-negative bacterium. 34. The process according to claim 33, characterized in that the gram negative bacterium is E. coli including a strain BL21 or a derivative thereof or a strain XA90 or a derivative thereof. 35. The process according to any of claims 1-34, characterized in that the cell is genetically modified to have an intracellular environment less reduced than an unmodified cell of the mi sm = strain. 36. The process according to any of claims 1-35, characterized in that the cell can be modified to have a lacking or reduced activity of a thioredoxin reductase or an enzyme having a similar effect on the potential that reduces the sulfhydryl of the cytoplasm. 37. The process according to any of claims 1-36, characterized in that the modified cell is a mutant TrxB. 38. The process according to any of claims 1-37, characterized in that the gene is a derivative of a cue gene occurring naturally. 39. The process according to claim 38, characterized in that the derivative is obtained by replacing at least one codon which is used more frequently by the host cell than one originally present where the codon codes for the same amino acid. 40. The process according to any of claims 1-39, characterized in that the gene is under control of a regulatory DNA sequence associated unnaturally with the gene. 41. The process according to any of claims 1-40, characterized in that the bacterial cell is transformed with a selected expression vector. • 5 from the group consisting of the pET vectors, for example, T7 promoters. 42. A stable peptide-free MHC protein, characterized in that it is obtained by a process in accordance with any of the 10 claims 1-41. • 43. A kit, characterized in that it comprises a class 1 MHC and a ß2m heavy chain allowing the recipient to produce and measure or detect a functional class 1 MHC protein 15 for a peptide, which is capable of binding to the MHC class 1 protein, can be added leading to the generation of a functional MHC class 1 protein. 44. The kit according to claim 43, characterized in that it comprises the labeling of one or more subunits of class 1 MHC (heavy chain, b2m and / or peptide) to measure or detect the generation of the class 1 protein MHC. ^ jyfgg ^^ 45. The kit according to claim 43 and 44, characterized in that the system for measuring or detecting the MHC class 1 protein generated is selected from the group of technologies consisting of radio-ligands, immuno-precipitation, ELISA, resonance. of plasma, fluorescent polarization, ul t racent ri analytical leakage, biochemical precipitation, ultrafiltration, chromatography and equil ibrio dialysis. 46. The kit according to claims 43-45, characterized in that it comprises an oligomerization of MHC proteins, such as two, three, four or more. 47. The kit according to claims 43-46, characterized in that an additional reagent is added as a marker making the kit suitable for diagnostic purposes. 48-. The kit according to claims 44-47, characterized in that the marker is selected from the group consisting of biotin, f luorochromes, enzymes, chemoluminicente, and radioactive markers. 49. The use of a process according to any one of claims 1-41, in the manufacture of MHC. 50. The use of stable empty MHC protein according to claim 42, in the analysis of the effect of changing an amino acid in the MHC on the binding specificity of the MHC as evaluated by an analysis using a peptide library proximate to be a synthetic or recombinant collection. METHOD FOR PRODUCING A FUNCTIONAL PROTEIN OF THE IMMUNOGLOBULINES SUPERFAMILY SUMMARY OF THE INVENTION The present invention relates to a process for producing a functional protein of the immunoglobulin superfamily, which has at least one disulfide bond when it is functional, the process comprises the steps of providing a bacterial cell comprising a gene coding for the protein, the gene is able to be expressed in the cell, cultivate the cell under conditions where the gene is expressed, isolate the protein from the cell without reducing it, and subject the isolated protein to a bending treatment. Preferably the protein of the immunoglobulin superfamily is selected from the group consisting of antibodies, variable (V) immunoglobulin regions, immunoglobulin constant C regions, immunoglobulin light chains, immunoglobulin heavy chains, CD1, CD2, CD3, molecules histocompatibility class I and class II, Beta2microglobulin (ß2m), antigen-3 associated in lymphocyte function (LFA-3) and Fc? RIII, CD7, CD8, Thy-1 and Tp44 (CD28), T cell receptor, CD4, poly-immunoglobulin receptor, neuronal cell adhesion molecule (NCAM), myelin-associated glycoprotein (MAG), myelin P protein, carcino-embryonic antigen (CEA), platelet-derived growth factor receptor (PDGFR), receptor of colony stimulation factor-1, alpha beta-glycoprotein, ICAM (intercellular adhesion molecule), platelets and interleukins. Important embodiments of the invention is a peptide-free stable MHC protein, obtainable by a process of the invention and a kit comprising a MHC class I heavy chain and a (ß2m) that allows the recipient to produce and measure or detect an MHC protein Functional class I to which a peptide, which is capable of binding to the MHC class I protein, can be added to drive the? -nection of a functional MHC class I protein.