WO2003097669A2 - Method for purifying denatured proteins having a desired disulfide bond configuration - Google Patents

Abstract

The present invention relates to a method for production of a protein having a desired fold. This is especially achieved by subjecting a population of proteins to a separation step under non-reducing conditions. This allows for identification of a sub-population of proteins having the disulfide bond configuration resulting in a desired fold. Most often this will be the protein of proper structure and/or function. Thus, by using the novel method the purity of the protein having a desired fold can be increased as compared to the purity of a similar protein produced by a conventional method. Important aspect of the invention is a functional active MHC heavy chain protein obtainable by the above method and the use of a MHC heavy chain protein in analysis of peptide binding capacity.

Description

A METHOD FOR PURIFYING DENATURED PROTEINS HAVING A DESIRED DISULFIDE BOND CONFIGURATION

FIELD OF INVENTION

The present invention relates in its broadest aspect to the field of protein biochemistry in particular folding of a protein from a suspension comprising the protein in an essentially unfolded and thus inactive form. More specifically, there is provided a novel method for production of a protein having a desired fold. This is especially achieved by subjecting a population of proteins to a separation step under non-reducing conditions. This allows for identification of a sub-population of proteins having the disulphide bond configuration resulting in a desired fold. Most often this will be the protein of proper structure and/or function. Thus, by using this novel method the purity of the protein having a desired fold can be increased as compared to the purity of a similar protein produced by a conventional method. Folding of the protein is initiated from a purified denatured protein containing the correct disulphide bond configuration, accordingly, the folding efficiency and rate can be increased considerably as compared to conventional methods, resulting in a increased yield of the entire production process.

BACKGROUND OF THE INVENTION

Formation of disulphide bonds is an essential co- and post-translational event in the folding reaction of many secretory proteins. In several instances this particular event has been shown to be the rate-limiting step in the folding reaction pathway leading from the unfolded state to the properly folded and functional state of the protein. The number of possible disulphide bond isomers that can be formed during folding increases exponentially with the number of cysteine residues in the protein. Generation of stable and/or semi- stable incorrect disulphide bond isomers during folding not only decrease the yield and efficiency of the folding reaction but also the purification process. The latter is due to the fact that disulphide bond isomers, especially under denaturing condition, but also under non-denaturing conditions, resemble each other structurally, making it extremely difficult to separate the isomers using conventional purification methods, which exploit molecular differences such as size, hydrophobicity, charge etc. to achieve the separation.

Conventional methods, within the field of production and folding of disulphide containing proteins, most often relate to methods were formation of disulphide bonds is performed during the folding treatment. Further, the formation of disulphide bonds typically involve extraction of aggregated protein under reducing conditions, hence, the subsequent folding treatment is initiated from the denatured and fully reduced state. This reaction is exceedingly slow and inefficient, as the molecule has to go through a high number of possible intermediate stages before reaching the properly folded state. To compensate for this, additives such as redox pairs and/or disulphide bond isomerases (a specific sub-group of proteins that catalyse the formation and/or shuffling of disulphide bonds) are added to the folding reaction to control and promote disulphide bond formation (under in vitro conditions, to mimic the situation under in vivo conditions). However, addition of the above listed compounds does not solve the above described problem of generation of incorrect disulphide bond isomers during folding as the formation of these isomers can still not be avoided. Furthermore, the addition of such additives increases the cost of the folding reaction considerably.

Current methods for folding of proteins does not take these considerations into account.

A method of producing proteins under conditions which do not change the disulphide bonds generated by the cell is disclosed in WO 00/15665. This patent application 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. The application does not relate to the existence of isomers and as a result the folding treatment is initiated from a population containing a mixture of isomers.

Thus, all current strategies for folding and/or production of proteins having a desired fold fail to recognise the potential of forming the correct disulphide bonds and purifying the proteins comprising these correct disulphide bonds to homogeneity using a separation technique under denaturing and non-reducing conditions and exploiting the possible advantages of disulphide bond assisted folding.

BRIEF DESCRIPTION OF THE INVENTION

The present invention pertains in one aspect to a method of purifying, from a population of different disulphide bonded isoforms of the same monomeric protein, the isoform(s) of optimal refolding ability, the method comprising the steps of (i) solubilising the population by adding a denaturant, (ii) subjecting said solubilised population to at least one separation step, under denaturing and non-reducing conditions, resulting in the separation of at least one isoform, (iii) obtaining the isoform(s) of optimal refolding ability, and (iv) subjecting the purified isoform(s) to a refolding treatment.

In further aspects the invention provides a functionally active MHC heavy chain protein obtainable by the above method.

In a still further aspect, the invention relates to the use of the above MHC heavy chain protein in analysis of peptide binding capacity.

DETAILED DISCLOSURE OF THE INVENTION

Factors affecting protein folding include hydrophobic interactions, hydrogen bonding and ionic interactions. An essential part of protein folding is the formation of disulphide bonds and in many cases this particular event has been shown to be the rate-limiting step in the folding pathway.

The number of possible disulphide bond configuration increases exponentially with the number of cysteine residues present in the protein structure. A protein comprising four cysteine residues forming two disulphide bonds give rise to 10 possible disulphide bond configurations, one fully reduced, six partially oxidised and three fully oxidised. Therefore, identification and separation of a sub-population of proteins with the correct disulphide bonds i.e. the disulphide bonded isomer(s) of optimal refolding ability will inevitability result in an increased purity of the protein. The preparative separation of monomeric disulphide bond isomers (i.e. comprising correct and in-correct configurations) under denaturing and non-reducing conditions prior to the refolding treatment, using any separation method available, has not been described in the scientific literature. Consequently, the folding reaction of conventional methods are always initiated from a population of proteins containing a mixture of correct and in-correct disulphide bond isomers.

Accordingly, all proteins comprising cysteine residues will gain from being produced in accordance with the method of the present invention. The person of skill in the art being introduced to the principle of the present invention will immediately realise the potential of the method according to the present invention. The efficiency of obtaining isoform(s) of optimal refolding ability and the purity of the resulting protein will represent obvious improvements in the production as compared to conventional methods. Additionally, the possibility of separating one or more sub-populations (isoforms) of the desired protein opens for the opportunity to discover new and previously unknown characteristics attributed to these specific sub-populations or isomer forms of the desired protein.

In the following, the term "disulphide bond isomer", "isomer form" and "isomer" are used interchangeably to denote the possible sub-populations that can be derived from a population of the same monomeric protein.

The term "same monomeric protein" refers to a protein structure composed of a single polypeptide chain i.e. an amino acid chain of the same length and sequence. It should however be understood, that the present invention is not limited to proteins composed of only one polypeptide chain as the method could also be employed to larger macromolecular structures, in which several monomeric protein structures are held together through non-covalent bonds, such as hydrophobic, hydrophilic, van der Waals, hydrogen and ionic/salt bonds.

The expression "protein having a desired fold", "isoform of optimal refolding ability" and "correct disulphide bond" are used in order to characterise the protein having the disulphide bond configuration, which will be regarded as the desired protein; in most cases this will be the disulfide bond configuration found in the native protein.

The present invention relates to a process of purifying such a protein having a desired fold. This is achieved by identifying, in a population of said protein, at least one sub-population of proteins with correct disulphide bonds and obtaining at least one sub-population of optimal refolding ability. The proteins comprising the correct disulphide bonds represent the sub-population of proteins having a desired fold. Typically, the desired protein is a protein of proper biological functionality, structure and/or function. In the present context the protein having a desired fold may also be referred to as the "protein of interest" or "the desired protein". It is appreciated that the desired protein is a protein having a characteristic that is desired. Such characteristics include proper biological functionality, structure and/or function as described above and may in addition include characteristics such as stability, allergenicity, solubility, charge, size and shape. In a specific embodiment of the method of the present invention, it may be of interest to obtain a sub-population of proteins that are not generally regarded as the protein of interest i.e. of proper biological functionality, structure and/or function. In the production of proteins e.g. for medical uses it may be desirable to obtain a particular isomer form of a protein which has specific characteristics such as e.g. reduced allergenicity and/or reduced or increased stability. Accordingly, the expression "isoform(s) of optimal refolding ability" when used in the present context covers all possible isoforms of the same monomeric protein which can be of interest. The characteristics of the protein having a desired fold may, as indicated above, be very variable and dependent on the particular protein of interest. In the present context, the term "protein having a desired fold" designates a protein with correct disulphide bonds as defined above with a structure and/or function allowing said protein to perform at least one of the functions attributed to said protein at least to a substantial degree e.g. as assessed by an in vitro assay and/or having any other characteristic which can be defined using standard methods known in the art.

In a preferred embodiment the protein to be purified according to the method of the present invention comprises at least two cysteine residues. The protein may comprise at least 4, such as at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 18, or at least 20 cysteine residues. Preferably, the protein comprises an even number of cysteine residues. Most likely, the protein is having at most 20, such as at most 14, at most 10, at most 8, at most 5, at most 4, at most 3, or at most 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 5, or at least 6 disulphide bonds. Preferably, the protein comprises no unpaired cysteine residues. However, within the scope of the invention is an embodiment wherein the protein comprises 1 or more unpaired cysteine residues.

Proteins to be purified according to the method of the invention include all natural and synthetically produced proteins.

The present invention is exemplified with reference to the MHC class I heavy chain protein. However, as mentioned previously, all proteins with the potential of giving rise to different disulphide bonded isoforms may be purified, and thus produced, according to the method of the invention. Such proteins include e.g. a protein of the immunoglobulin superfamily i.e. a protein 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 histocompatibility molecules, β₂microglobulin (β₂m), lymphocyte function associated antigen-3 (LFA-3) and FcγRIII, 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, carcinoembryonic antigen (CEA), platelet derived growth factor receptor (PDGFR), colony stimulating factor-1 receptor, β-glycoprotein, ICAM (intercellular adhesion molecule), platelet and interleukins.

More specifically, a protein to be produced according to the method of the invention is a protein selected from the group consisting of proteins comprising a heavy chain, a heavy chain combined with a β₂m, a functional mature MHC class I protein, and a MHC class II protein selected from the group consisting of an α/β dimer and an α/β dimer with a peptide.

In a specific embodiment of the method of the invention the protein to be produced is a MHC class I protein, preferably a human MHC. The produced MHC protein may be obtained as a peptide free MHC protein. By using the method of the present invention pure MHC class I heavy chain monomers can be obtained and accordingly perform highly efficient folding in the presence of β₂m and a specific peptide.

Thus, the origin of the protein to be produced according to the method of the invention may be eukaryotic as well as prokaryotic. The eukaryotic proteins include proteins derived from a vertebrate species selected from the group consisting of humans, a murine species, a rat species, a porcine species, a bovine species and an avian species.

Furthermore, the protein to be purified may be derived from recombinant protein expression in transformed host organisms or cell lines. Useful prokaryotic cells for expression can be selected from Gram negative and Gram positive bacteria. Examples of useful Gram negative expression cells include Enterobacteriaceae species such as e.g. Escherichia spp. Salmonella spp. and Serratia spp; Pseudomonadanaceae species such as Pseudomonas spp., and examples of Gram positive bacteria that can be used in the invention include Bacillus spp., Streptomyces spp and lactic acid bacterial species. Suitable eukaryotic cells for expression can be selected from fungal cells including yeast cells, mammalian cells including human cells and insect cells.

Recombinant protein expression often results in the formation of insoluble aggregates and it is to be understood that the protein to be purified according to the method of the invention can be part of any structure selected from the group consisting of inclusion bodies, aggregates, insoluble complexes, intermolecular complexes and intramolecular complexes. The tendency to form insoluble aggregates does not correlate with protein characteristics such as the size of the expressed polypeptide, the use of fusion constructs, the subunit structure, or the relative hydrophobicity of the recombinant protein. Overproduction by itself is frequently sufficient to induce the formation of inactive aggregates. Studies of recombinant protein expression in e.g. Escherichia coil have shown that inclusion body formation is a very common phenomenon.

In accordance with the method of the invention a population of solubilised proteins are provided by adding a denaturant. The term "population" is used to denote an accumulation of the protein of interest comprising all its possible isomer forms. The protein of interest will most often be present in a mixture with contaminants such as e.g. other proteins, cell debris, lipids, carbohydrates, DNA and RNA. It is contemplated that the protein of interest not necessarily constitutes a major part of the mixture. Accordingly, it may be preferred to remove such contaminants by any suitable method known in the art. The method of removing contaminants may be applied at any stage during the method as described in the present invention. Preferably, contaminants are removed prior to folding treatment.

Methods for removing contaminants are described in the art and include chromatographic methods such as ion-exchange, hydrophobic interaction chromatography, partition chromatography, including reversed phase liquid chromatography, adsorption chromatography, expanded bed adsorption chromatography, high gradient magnetic fishing, affinity chromatography, including fusion protein chromatography, high pressure liquid chromatography (HPLC), size exclusion chromatography and centrifugation techniques, including density gradient separations.

It is further appreciated that the population of protein is provided in a solubilised form. A "solubilised form" include proteins which may be obtained in a solubilised folded form expressed from a microorganism, such as fungi or yeast, capable of handling correct folding of the protein. In situations as described above where the proteins are expressed in a substantially mis-folded form it is appreciated that the proteins are solubilised and unfolded in a medium by treating the proteins with a substance that can keep the proteins in a substantially unfolded form including random coils. Such media include denaturants typically selected from the group consisting of organic solvents such as ethanol and propanol; chaotrophic agents such as urea, guanidin hydrochloride, thiocyanate; detergents such as sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide

(CTAB) or deoxycholate; salts such as KSCN or LiBr. The concentration of the chaotrophic agent such as urea may be in the range of 0,5-9 M such as in the range of 5-7 M, in particular about 8 M.

It may be beneficial to add, to the media comprising the solubilised protein, an agent which inhibits proteolysis. Useful proteolysis inhibitors are described in the art and include compounds selected from the group consisting of cysteine, aspartic acid, serine, metallo proteinase inhibitors such as N-ethyl-maleimide, pepstatin, phenyl methyl sulphonic flouride (PMSF) and EDTA, respectively, and of ATP dependent proteolysis inhibitors such as sodium ortho vanadate.

The proteins may be solubilised by denaturation under non-reducing conditions, optionally without altering the redox state. Alternatively, the denaturing step may be performed under reducing conditions. Examples of useful reductants include compounds selected from the group consisting of dithiothreitol, dithioerytritol, gluthathione, cysteine, cystamine and 2-mercaptoethanol.

In a specific embodiment, it may be advantageous to adjust the redox potential of the solution containing the protein of interest, optionally by using a reductant and/or oxidant. Useful redox pairs may be selected from the group consisting of reduced glutathione (GSH)/oxidized glutathione (GSSG); cystamine/cysteamine; reduced dithiothreitol (DTTred)/oxidized dithiothreitol (DTTox) or other redox pairs known to the person skilled in the art. Furthermore, it may be preferred to adjust the redox potential during the folding treatment in a similar manner.

In particular embodiments the reduced denatured proteins are oxidised under denaturing conditions by adding an oxidant including but not limited to the oxidants listed above. This secures the possibility of pre-forming/locking the disulphide bond configurations which distinguish the disulphide bond configuration of the desired protein from other isomer forms having incorrect disulphide bond configurations. Hence, the desired protein can be separated.

An essential feature of the method of the present invention is to subject the population of solubilised proteins to at least one separation step resulting in identification/separation of at least one sub-population of proteins. The separation step is performed under denaturing and non-reducing conditions, to ensure that the proteins are soluble during the purification step and that the redox state is not changes once the correct disulphide bond configurations have been formed. The outcome of this separation step is the purification of denatured molecules having disulfide bond configuration(s), which optimally facilitates refolding upon removal of the denaturant. This subpopulation(s) of molecules are referred to as "isoform(s) of optimal refolding ability" as described above.

Thus, The principle of the present method can be applied in at least 2 different production scenarios. Scenario 1 : Aggregated protein is solubilised by extraction into a denaturing agent under non-reducing conditions and the sub-population of interest is identified and subsequently separated from other sub-populations by a separation method. The sub- population is then subjected to a renaturation treatment. Scenario 2: Aggregated protein is solubilised by extraction into a denaturing agent under reducing conditions and then oxidized under denaturing conditions. The sub-population of interest is identified and separated from other sub-populations. The sub-population of interest is then renatured.

The inventors of the present method have shown that it is possible to separate isomer forms of a protein and obtain the sub-population representing the protein having a desired fold. Separation as well as identification/purification techniques are described in the art. The person of skill within the technical field relating to protein separation and purification will be able to select methods that will perform the separation of sub-populations of the protein of interest. Useful techniques include, but are not limited to, dialysis, filtration, dia- filtration, tangential flow-filtration, gel-filtration, extraction (two-phase extraction). precipitation, centrifugation, electrophoretic techniques and chromatographic techniques.

In a preferred embodiment, a chromatographic technique is used for separation and/or purification of the sub-population of the desired protein. Such techniques include ion- exchange chromatography, reverse phase chromatography, hydrophobic interaction chromatography, affinity chromatography and mixed mode chromatography. For purification of a target molecule, the specific sub-population, the techniques are based on characteristics of the target molecule such as solubility, charge, size, shape or affinity, which cause the target molecule to be captured/retained due to interactions/reactions.

Accordingly, the separation step may be performed using any present or future separation method which allows separation of the isomer forms of the desired protein.

In a presently preferred embodiment of the method of the invention hydrophobic interaction chromatography is used in the separation step. This technique can benefit in separation of the protein of interest when being applied in combination with ion exchange chromatography and size exclusion chromatography. A combination of chromatographic methods that utilise different protein properties, such as charge and size, to achieve a given separation of a population of proteins is usually more effective than applying different chromatographic techniques that exploit the same protein property, such as an anion-exchange step followed by another but slightly different anion-exchange step.

In a further embodiment separation and accordingly identification of the sub-population of proteins in accordance with the method of the invention is performed using a method selected from the group consisting of electrophoretic techniques such as SDS-PAGE, electro-blotting (ex. western blotting), capillary electrophoresis, isoelectric focusing and mass spectroscopy, HPLC (high pressure liquid chromatography), LS (liquid chromatography), and GC (gas chromatography).

In a presently preferred embodiment, the method for identification of sub-populations is SDS-PAGE.

In accordance with the method of the invention, the obtained sub-populations of proteins with correct disulphide bonds are subjected to a folding treatment. Methods for folding of proteins are described in the art. Folding which can also be referred to as renaturation is typically performed by dilution or dialysis. Upon removal of the denaturing or chaotrophic agent, the protein is exposed to intermediate denaturing concentrations, allowing the protein to fold spontaneously.

Suitable folding buffers are characterised as fluids allowing the protein to refold. Such buffers are described in the art and include TrisHCI buffer and EDTA. It may be preferred to make a buffer system by including a suitable additive to the buffer system and selecting the proper pH and ionic strength of the buffer system. A buffer system for folding of the protein in question may easily be designed by the person skilled in the art.

Conditions which may influence the folding of proteins are described in the art and include physical parameters such as e.g. volume, flow of reactants and buffers, temperature and pressure; chemical parameters including pH, ionic strength, reduction potential, oxidation potential, detergents, protease inhibitors and ATPase inhibitors and enzymatic parameters including heat-shock proteins, oxidating or reducting enzymes and disulfide isomerases. In a specific embodiment of the method according to the invention the folding treatment is performed essentially in the absence of reducing agents. However, folding may be performed in the presence of a reducing agent which will affect reduction of inappropriate disulphide bonds without affecting appropriate disulphide bonds.

As described above the folding treatment is typically performed after the proteins have been subjected to the separation step. Obtaining the protein isomer with the correct disulphide bond configuration before subjecting the proteins to a folding treatment ensures a faster and more efficient folding process. Indeed, the inventors of the present method have surprisingly shown that it is possible to separate the MHC heavy chain isomer comprising the correct disulphide bond configuration from the incorrect isomers under denaturing conditions using the described method. It is, however, contemplated that identification and subsequent obtaining of the protein isomer with the correct disulphide bond configuration can also be performed after the protein has been subjected to a folding treatment. In this particular embodiment the separation step ensures a considerable increase in the purity of the final protein preparation.

Furthermore, it is well known that formation of disulphide bonds are favoured under slightly alkaline conditions typically around pH 8. However, as the correct disulphide bonds are formed prior to the folding treatment, this treatment can be performed at pH values which are not necessarily optimal for disulphide bond formation as these are already formed. Accordingly, folding can be performed at pH values less than or equal to pH 7. An example is the peptide binding to the MHC class I molecule which is optimal at pH 6.6. It is a highly interesting feature of the present invention that the protein having a desired folding is obtained at a higher purity, higher yield, higher recovery and at a faster rate as compared to a similar protein produced using conventional methods. As illustrated in Fig. 1 the energy requirement for folding of a protein using the method of the present invention is considerably reduced as compared to a similar protein produced according to conventional methods. Thus, the efficiency of a renaturation treatment performed on proteins produced in accordance with the present method will be increased when comparing to a protein produced according to conventional methods.

The principle of the present invention is further outlined in the Fig. 2. The figure illustrates the relative distribution of proteins with correct disulfide bond configuration (white), incorrect disulfide bond configuration (spotted) and contaminants (black) using a conventional method (top) or the method of the present invention (bottom). The figure clearly demonstrates that extraction under reducing conditions followed by re-oxidization and separation under controlled conditions results in a higher yield an improved purity of the protein of interest when the method of the present invention is applied.

Yield or recovery may be defined as the total activity at a given step during the isolation of the protein having a desired fold divided by the total activity at a reference step e.g activity of the solubilised protein (step (i)). The person of skill in the art will be able to select a method for determining yield and recovery using suitable method for determining e.g. the peptide binding or the enzymatic activity of the protein of interest.

Activity may be defined as the measure used to evaluate/calculate/determine the yield/recovery of the desired protein in a single step in the production, folding treatment and/or the purification or as the total yield of the entire process resulting in the protein having a desired fold.

Further, activity may be defined as the measure of the proteins ability to obtain and/or carry out the assigned structure and/or function under a defined set of conditions. The activity of the protein of interest can be measured using an appropriate assay technique such as ELISA, spectrophotometric methods including visible, ultraviolet and luminescence, spectrofluorimetric methods, protein-ligand binding studies, radioimmunoassay including radiolabelled peptide binding to MHC class I molecules.

In situations where the desired protein is an enzyme the specific activity at any step during production of the desired protein may be provided as the number of activity units per unit of total protein and used in determining the yield. Obviously, the assay used for determining yield or recovery must be selected to match the desired protein.

Furthermore, the protein produced according to the present invention is obtained at purity which is considerably increased.

In the present context, purity is to be understood as a preparation in which all proteins are essentially those of a single form e.g disulphide bond isomer. This allows for an efficient folding treatment and at the same time allows for efficient removal of any contaminating matter, as illustrated in Fig. 2.

By using the method of the present invention for purification of a protein having a desired fold it is possible to obtain the protein at a high purity. Preferably, it is possible to obtain a protein produced in accordance with the method of the present invention that is more than 70% pure, such as at least 80%, e.g. at least 85, such as at least 90%, e.g. at least 93%, preferably at least 95% or even at least 99% pure.

The specific values of purity and or yield and discovery may vary considerably depending on the protein to be produced. In the method according to the present invention the total yield of properly folded protein is at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95%. The total yield may be at least 10 mg folded protein, including at least 100 mg folded protein, such as at least 1 g folded protein, 10 g folded protein, 100 g folded protein, including at least 1 kg folded protein, 10 kg folded protein, 100 kg folded protein, or even under large scale folding at least 1 t of properly folded protein.

A most important aspect of the invention is a functionally active MHC heavy chain protein which is obtainable by a method according to the invention. A further important and highly useful aspect of the present invention is the use of the above MHC heavy chain protein in analysis of peptide binding capacity. Such a functional MHC and use of the protein are described in the following. It is contemplated that the principle of the present invention can be used to provide any protein having a desired fold. Uses of such proteins are also within the scope of the present invention.

As mentioned before, the present invention is exemplified with reference to the MHC class I heavy chain. In the following the specific advantages of producing this particular protein in accordance with the method of the present method is described.

The major histocompatibility complex class I (MHC-I) molecules are expressed on the surface of almost all cells in the body. These molecules are ternary complexes consisting of 1) a glycosylated heavy chain (44 kDa), containing two disulfide bonds, and 2) a noncovalently associated light chain, b2-microglobulin (12 kDa), containing a single disulfide bond, and 3) a tightly bound peptide (Springer et al., 1979). Their function is to sample endogenously derived peptides, transport them to the cell surface and present them to cytotoxic T cells, which continuously scan cell surfaces for peptide-MHC-I complexes. Peptides presented in context with MHC-I molecules originate from the digestion of intracellular proteins, normal ones as well as those of pathogens. MHC-I molecules therefore serve as a link between the intracellular compartment, which is inaccessible to the cells of the immune system, and the extracellular compartment, where the immune cells reside. This mechanism is crucial in the immune defense against intracellular pathogens, such as viruses.

Understanding the specificity of MHC molecules, and, in particular, the rules defining whether a peptide will be bound or not, is therefore of considerable interest to the immunologist. Quantitative peptide-MHC-I binding kinetics can be determined from biochemical binding experiments and artificial neutral networks can trained to predict binding of unknown peptides (reviewed in Buus, 1999). MHC molecules are not only the target of extensive research, but also an important scientific tool for identification, characterisation and stimulation of specific subsets of T cells. The methodology is based on the increased avidity of tetrameric MHC-peptide complexes for the T cell receptor (TCR) (Altman et al., 1996). Generating tetrameric MHC peptide complexes and/or studying MHC-peptide interactions relies heavily on the availability of highly pure and fully functional MHC molecules.

Purification of soluble peptide-MHC complexes from human cell lines, using monoclonal antibodies, is a cumbersome and expensive process resulting in low protein yields. Expression of MHC-I molecules in bacterial hosts offers significant advantages including ease of genetic manipulation and reasonably simple growth and induction methodologies leading to high yield and purity. However, high levels of expression frequently lead to the formation of insoluble inclusion bodies and this phenomena has been shown to be the case for expression of the heavy chain and β₂m in E. coli (Garboczi et al., 1992). To regain the biological activity of the molecules, extraction into denaturing buffer followed by in vitro protein refolding is necessary. The refolding process is complicated by the fact that the heavy chain cannot fold to its native state in the absence of β₂m and peptide. So far, it has been impossible to generate empty MHC-I molecules (i.e. not preoccupied with peptide), as they are highly unstable and prone to aggregation unless peptide is bound. Another strategy to obtain high quality MHC-I molecules would be to express and purify the two polypeptide parts separately and then initiate folding by adding them together with a specific peptide (Garboczi et al., 1992). The final reconstitution step could then be used as a peptide binding assay, allowing any conceivable combination of heavy chain and peptide. MHC-I molecules generated in this way could then be used for T cell analysis and generation of tetrameric peptide-MHC-I complexes. To obtain the increased avidity of these molecules for the TCR it is essential that all peptide-MHC-I molecules constituting the tetrameric complex are fully functional and loaded with the same peptide.

The key to high yields of functional MHC-I molecules lies in the refolding step. As the molecules contain several disulfide bonds refolding must be performed under alkaline conditions, typically around pH 8.0 (Rudolph et al., 1996), to promote disulfide bond formation. However, it has been shown that peptide binding, which is necessary for stable complex formation, is optimal at acidic pH, around pH 6.6. This poses a serious problem, as disulfide bond formation and peptide binding cannot be optimized simultaneously. A possible solution to the above conundrum lies in uncoupling these two events from one another so that each of them can be optimized independently.

In the following examples, it will be demonstrated that disulfide bond isomers of the heavy chain molecules can be separated by hydrophobic interaction chromatography under non- reducing, denaturing conditions and that pure preparations of oxidized MHC-I heavy chain molecules can be obtained. The active isomer is subsequently identified and shown to undergo essentially complete disulfide assisted refolding under conditions optimized for peptide binding.

The invention is further illustrated in the following non-limiting examples and in the figures:

Figure 1:

Energy landscape describing the possible pathways towards the properly folded state for proteins produced using conventional methods and for proteins produced in accordance with the present method. Where the number of pathways are multiple the likelihood of the protein becoming trapped in a semi-stable state along the way increases and as result the folding reaction is slow and inefficient. The possible pathways towards the properly folded, native state is significantly reduced when the protein is oxidized and separated under denaturing conditions prior to the renaturation step according to the method of the present invention. This leads to a more efficient and faster renaturation process as the molecule is already locked in a favorable conformation for renaturation. Thus, by using the present method the protein having a desired fold can be obtained fast and at a high purity.

Figure 2: Relative distribution of proteins with correct disulfide bond configuration (white), incorrect disulfide bond configuration (spotted) and contaminants (black) using a conventional method (top) or the method of the present invention (bottom). After solubilisation of inclusion bodies (A, 1), purification (2, 3, 4) and separation (B, D) and re-oxidization (C) under reducing (top) and non-reducing (bottom) conditions. The height of each individual pie indicates the total amount of protein present after each step. In the production scheme according to the method of the present invention the protein of interest is obtained in an oxidized state, of which a considerable part will have obtained the correct disulphide bond configuration. The essential step in the invention is the following removal of protein isomers with in-correct disulphide bond configuration by e.g. hydrophobic interaction chromatography under denaturing and non-reducing conditions. The folding treatment is then performed on the pure denatured protein isomer with the correct disulphide bond configuration. In contrast, the conventional process dictates the application of the folding treatment on the fully reduced denatured protein, leading to lower folding efficiency and yield. Furthermore, the present invention ensures a considerable increase in the final purity of the protein product as protein isomer with in-correct disulphide bonds can be removed.

Figure 3:

Reducing SDS-PAGE analysis of expression levels of MHC-I heavy chains in E. coli. A: Expression levels of rA2. B: Expression levels of rKk (des cys). C: Expression levels of rAll. Fermentor samples (15 μl) were withdrawn before and every hour after induction.

The bacterial cell pellet was resuspended in 50 μl MgCI2/SDS lysis buffer to release and solubilize heavy chain inclusion bodies as described by Chen & Christen, (1997). After centrifugation at 20,000 g for 2 min, 15 μl of the supernatant was loaded directly on the gel. Lanes: 1: Protein marker; 2: Before induction; 3-5: Samples taken 1, 2 and 3 h after induction with IPTG, respectively. Positions of heavy chain monomers are shown with arrows.

Figure 4: Reducing and non-reducing SDS-PAGE analysis of solubilized inclusion body preparations. A: Solubilized inclusion bodies containing rA2. B: Solubilized inclusion bodies containing rAll. The intensity of the heavy chain monomer band increased upon reduction, due to the release of disulfide bond cross-linked monomers, and made the distinction of the isomers difficult. To improve the visualisation of heavy chain isomers, reducing samples of rA2 and rAll were diluted twice as much in sample buffer as the non-reducing counterparts. The positions of heavy chain isomer 0, 1 and 2 are indicated on the figure. NR: Non-reducing, Red: Reducing.

Figure 5:

Separation of rA2 heavy chain isomers by hydrophobic interaction chromatography on Phenyl Sepharose High Performance under non-reducing denaturing conditions. Aliquots (7.5 μl) of fractions collected in the indicated area of the chromatogram were analysed by non-reducing SDS-PAGE and the result is shown below. Analysed fractions are indicated on the figure as well as the positions of heavy chain isomers. Lanes: M: Protein marker, L: Sample applied on the HIC column.

Figure 6:

Separation of rAll heavy chain isomers by hydrophobic interaction chromatography on Phenyl Sepharose High Performance under non-reducing denaturing conditions. Aliquots (12 μl) of fractions collected in the indicated area of the chromatogram were analysed by non-reducing SDS-PAGE and the result in shown below. Analysed fractions are indicated on the figure as well as the positions of heavy chain isomers. Lanes: M: Protein marker, L: Sample applied on the HIC column.

Figure 7:

Refolding and peptide binding analysis of fractionated MHC-I heavy chain isomers. A: Analysis of fractions collected during purification of rA2 on Phenyl Sepharose High Performance (see Fig. 3). B: Analysis of fractions collected during purification of rAll on Phenyl Sepharose High Performance (see Fig. 4). Folding was initiated by diluting aliquots (1 μl) from selected fractions 100-fold in 100 mM Tris-Maleate, pH 6.6 buffer, containing human β₂m (3 μM) and a radiolabeled peptide (15,000 cpm). The pixels intensities of heavy chain isomer 1 and 2 were calculated from a densitometric analysis of SDS- polyacrylamide gels shown in figure 3 and 4, respectively. Fraction numbers are shown on the figure. Symbols: '- Isomer 1 protein tracing, Ei : Isomer 2 protein tracing, gj: Mean peptide binding. The standard deviation of duplicate peptide binding measurements was typically within 5%.

Figure 8: Determination of the amount of oxidized rAll heavy chain monomer that refolds properly into the matured state with a quantitative and conformationally sensitive ELISA. The double log plot shows the amount of heavy chain monomer offered to the folding reaction that was detected in the fully matured MHC-I complex. A non-reducing SDS-PAGE analysis of the purified rAll sample is shown in the insert. Graded concentrations of denatured rAll were diluted in a 100 mM Tris-Maleate, pH 6.6 refolding buffer containing an excess of human β₂m (3 μM) and a specific peptide (10,000 nM). Detection of properly folded complexes and conversion of measured OD ₅₀ values to picomolar complex was performed as described in Materials and methods. Lanes: 1 : rAll sample, 2: Protein marker. Positions of rAll isomers 1 and 3 are shown with arrows. Standard deviations of triplicate measurements are indicated on the figure.

Figure 9:

Dose-response curve for purified rAll isomer 0 (reduced) and isomer 1 (oxidized). Graded concentrations of purified All isomer 0 and 1 were diluted 100-fold into 100 mM Tris- Maleate, pH 6.6 buffer containing human β₂m (3 μM) and a specific radiolabeled peptide (15,000 cpm) and 1 mg/ml Pluriol. The mean peptide binding values were calculated as described in Materials and methods. Symbols: ϋ : rAll isomer 1, j-B : rAll isomer 0. The standard deviation of duplicate peptide binding measurements was typically within 5%.

EXAMPLES

Materials and methods

Cloning of human and murine MHC-I-heavv chains

All recombinant MHC-I heavy chains were expressed without the transmembrane sequence. E. coli strain XA90 transformed with pHNl+ containing an HLA-A*0201 (rA2) heavy chain (1-275) cDNA insert was a kind gift from Drs. Wiley and Garboczi. cDNA segments encoding HLA-A*1101 (rAll) heavy chain (1-275) was PCR amplified and inserted into the pET28a+ vector (Novagen, Denmark). The rAll sequence was optimised for E. coli codon usage, using the QuickChange kit (Stratagene, USA) and appropriate primers. The plasmid was subsequently transformed into E. coli strain BL21- CodonPlus(DE3)-RP (Stratagene, USA). Clones, which produced protein upon induction with IPTG, were identified and the inserted sequence was verified by DNA sequencing (ABI310, Perkin Elmer, USA). Murine H2-K^k (des cys) cDNA containing 4 cysteines was inserted into the pET28a+ vector and transformed into BL21(DE3) (Pedersen et al., 2001). Briefly, cysteine 121, which is not involved in disulfide bond formation, was exchanged for arginine as found in other murine MHC-I molecules.

Expression of rA2, rAll, rK (des cys) in E. coli

Large-scale production was performed in a 2 I Labfors fermentor (Infors AG, Switzerland), according to the manufacturer's instructions. All fermentations were run with ECPM 1 media (Bernard & Payton, 2002). Primatone (20 % (w/v)) (Rode & Rode, Denmark) was used instead of casamino acids and 0.3 ml antifoam 289 (Sigma, Germany) was added to prevent foaming. The fermentor including media was autoclaved for 1 hour and appropriate sterile antibiotics were added after autoclavation. Inoculation cultures were set up in 200 ml YTχ2 medium containing 10 mM MgCI₂ and 0.5% (v/v) glucose plus the respective antibiotics. An inoculum, corresponding to 10 ml of an OD₆₀₀ = 1 culture, was transferred to the fermentor and expansion was done over night at 25°C - 28°C with atmospheric air as oxygen source. The growth rates were followed by measuring the OD₆₀₀ of samples withdrawn every hour. As soon as the atmospheric oxygen became limited the air source was changed to 100% oxygen. When the OD₆₀₀ reached 25, induction was initiated with 1 mM IPTG. At the same time the temperature was increased to 42 °C and additional sugar (87 % glycerol) was added in a fed batch mode. After 3 hours of induction the fermentor was harvested. Samples taken just before and during induction were kept for SDS-PAGE analysis.

Isolation and solubilization of inclusion bodies

The isolation and solubilisation procedure was performed essentially according to Maniatis et al., (1989). Cells lysis was done with lyzosyme (Sigma, Germany) and liberated DNA/RNA was digested with DNAse I (Sigma, Germany) and RNAse A (Sigma, Germany). Inclusion bodies were collected by centrifugation at 17,000 g for 10 min at 4°C and washed in PBS supplemented with 0.5 % (v/v) NP-40 (Sigma, Germany) and 0.1 % (w/v) deoxycholic acid (Sigma, Germany) followed by washing in 50 mM Tris-HCI, pH 8.0, 1 mM EDTA, 100 mM NaCI. Washed inclusion bodies were solubilised in 20 mM Tris-HCI, pH 8.0, 8 M Urea (200 ml for each 100 g of wet cell paste). Insoluble material was removed by centrifugation at 17,000 g for 15 min at 4°C. Supernatants were pooled and successively filtered through 8, 3, 1.2 and 0.45 μm filters and stored at -20°C until further processing.

Purification of MHC-I heavy chains

Chromatographic separations were performed at 12 °C on a AKTA prime workstation (Amersham Biosciences, Sweden), using a flow rate of 60 ml/h. All chromatographic media and columns were from Amersham Biosciences, Sweden, including 1 ml HiTrap columns for screening experiments. The purification was monitored by SDS-PAGE and bicinchoninic acid (BCA) assay (Pierce, USA). Urea decomposition leads to the generation of cyanate. To minimize the concentration of cyanate in Urea containing buffers, all solution were made freshly and used immediately, and the primary amine, Tris, was included as cyanate scavenger (Wingfield, 2002). A preparation of solubilised inclusion bodies was applied to a Q Sepharose Fast Flow column (2.6 x 65 cm) equilibrated with 20 mM Tris-HCI, pH 8.0, 8 M Urea (Merck, Germany) (loading buffer). Unbound material was washed off with 2 column volumes (CV) of loading buffer. Bound proteins were eluted with a linear 0 - 1 M NaCI gradient (5 CV). Fractions containing functional MHC-I heavy chain were pooled. Functional pools from Q Sepharose Fast Flow fractionation were adjusted to 100 mM Tris- HCl, pH 8.0, 8 M Urea and ammonium sulfate (Sigma, Germany) was added to 20 % (w/v) saturation. The solution was stirred at room temperature for 30 min and insoluble material was removed by centrifugation at 17,000 g for 10 min at 4 °C. The supernatant was filtered through a 0.45 μm filter and applied to a Phenyl Sepharose High Performance column (2.6 x 70 cm), equilibrated with 100 mM Tris-HCI, pH 8.0, 8 M Urea, 20 % ammonium sulfate. Unbound proteins were washed off with 1.5 CV of the same buffer. Bound proteins were eluted from the column with a linear 20 - 0 % ammonium sulfate gradient (5 CV). Prior to the subsequent size exclusion chromatography step, fractions were pooled and concentrated on a 10 kDa NMWL filter (Millipore, USA) in a stirred nitrogen pressure cell (Amicon, USA) to a final volume of 15 ml. Size exclusion chromatography was done on two Sephacryl 200-HR or two Sephacryl 400-HR columns (2.6 x 100 cm) connected in series. Columns were equilibrated with 20 mM Tris-HCI, pH 8.0, 8 M Urea or 20 mM Tris-HCI, pH 8.0, 6 M Guanidine hydrochloride. Purified heavy chains were pooled, aliquoted and stored at -20°C until further analysis.

Peptide radioiodination

Peptides were purchased from Schaefer-N, Denmark and purified to homogeneity by reverse phase HPLC chromatography, lyophilised and stored at -20°C. All preparations were quantified using the BCA assay. Radiolabeling was done with ¹²⁵Iodine (Amersham Biosciences, Sweden). Peptides used for refolding of MHC-I heavy chains and biochemical binding assays had the following sequences (in single letter code): FLPSDYFPSV for HLA- A*0201, KLFPPLYLR for HLA-A*1101 and SDYEGRLI (Influenza NP peptide₅o-₅₇) for H2-K (des cys).

Biochemical peptide binding assay Refolding conditions reported by Pedersen et al., (2001) for MHC-I heavy chain were used. Purified MHC-I heavy chain samples were refolded by 100-fold dilution in the presence of excess human β₂m (3 μM) and radiolabeled peptide (1-3 nM, 15,000 cpm/sample) for approximately 24 h at 18 °C in a total reaction volume of 100 μl per sample. The refolding buffer was 100 mM Tris-maleate buffer, pH 6.6 in PBS supplemented with 1 mg/ml pluronic copolymer Lutrol F-68 (BASF, Germany). The final concentration of Urea after dilution was 80 mM. The recombinant human β₂m was produced in our laboratory from E. coli fermentations (Pedersen et al., 2001). Binding of peptide to MHC-I heavy chains were measured by Sephadex G-50 spun column chromatography (Buus et al., 1995). The radioactivity of the excluded "void" volume, containing formed MHC-I complexes, and of the retained volume, containing unbound peptide, was measured by gamma spectrometry (Packard Instruments, USA). Peptide binding values were calculated by dividing excluded radioactivity with the total amount of radioactivity offered. Mean peptide binding values were obtained from duplicate spun column chromatography runs and expressed in percent.

Quantitative ELISA assay measuring peptide-MHC-I complex formation

Quantitative detection of peptide-MHC-I complex formation was performed as described by Sylvester-Hvid et al., (2002). In summary, peptide-MHC-I complex formation was measured with a sandwich ELISA using the conformationally sensitive monoclonal antibody W6/32 for capturing complexes and horseradish peroxidase-conjugated, polyclonal rabbit anti-human β₂m antibody (DAKO, Denmark) for detection of correctly folded complexes. The ELISA was developed with 3,3' 5,5' -tetramethylbenzidine hydrogenperoxide (TMB- one, Kem-En-Tec, Denmark) for 30 min at room temperature and the color reaction was read at 450 nm on a Victor²Multilabel ELISA counter (Wallac, Finland). A standard curve was constructed by plotting the measured OD₄₅₀ response against the logarithm of an MHC standard with known protein concentration. The curve was optimally fitted to a sigmoid curve (Prism® 3.0, GraphPad, USA), thereby allowing OD₄₅₀ of any sample to be converted to the concentration of MHC-I complexes in the sample.

Electrophoresis

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli, (1970), using 1 mm thick mini gels containing 12 % polyacrylamide resolving gels (w/v) and 5 % (w/v) stacking gels. SDS-PAGE analysis of samples taken during fermentation was done according to the procedure described by Chen & Christen, (1997). Protein bands were visualized with Coomassie Brilliant Blue. Protein standards (SDS-7) were from Sigma, Germany. Densitometric analysis was performed on non-reducing, Coomassie blue stained gels, using an ID Image Analysis Program (Kodak, USA).

RESULTS

Expression of MHC-I heavy chains in E.coli

Membrane truncated recombinant HLA-A*0201 (rA2), HLA-A*1101 (rAll), and H2-K^k (rK^k (des cys)) were produced as insoluble inclusion bodies in E. coli. Expression was initiated with IPTG and allowed to continue for 3 hours at 42 °C. Low levels of expression were reached approximately 1 hour after addition of IPTG for both rA2 and rK^k (des cys) (Fig. 3A, B). In contrast the expression of rAll reached much higher levels, which were attained after 3 hours of induction (Fig. 3C). The observed difference in the expression levels are probably due to the fact that the gene encoding rAll was optimized for E. coli codon usage, which was not the case for rA2 and rK^k (des cys).

Isolation and solubilization of inclusion bodies

Inclusion bodies were released from harvested E. coli cells by enzymatic disruption with lysozyme and co-released DNA/RNA was subsequently digested with DNAse I and RNAse A to reduce the viscosity. Centrifugally collected inclusion bodies were then washed free of loosely adsorbed and entrained contaminants in several washing steps, before solubilization in 8 M urea under non-reducing conditions. A prerequisite for the chosen strategy is that inclusion body solubilisation must be performed under non-reducing conditions so as to preserve the oxidation state of the heavy chain molecules. The initial purities were estimated to be 20-30% by densitometric analysis of non-reducing SDS- polyacrylamide gels and total inclusion body protein recovery was approximately 0.3 - 1.5 g/l bacterial culture. Final OD₆₀₀ values of the fermentor culture prior to harvest were in the range of 30-60.

Table 1. Summary of purification steps for rA2, rAll and rK (des cys) rA2 rAll rK^k (d( as cys)

Purity of Purity of Purity of

Pool Total Pool Total Pool Total

Purification step isomer Yield isomer Yield isomer Yield volume protein volume protein volume protein

I^s (%) l³ (%) l^a (%)

(ml) (mg) (ml) (mg) (ml) (mg)

(%) (%) (%)

Solubilized IB 180 624 25 100 200 2265 34 100 175 1940 22 100

Q Sepharose Fast Flow 150 392 36 90 180 652 39 33" 235 832 41 78

(NH₄)₂S0₄ precipitation 160 42 43 46 190 218 37 10 245 71 87 14

Phenyl Sepharose H^c 200 38 92 23 200 72 90 8 280 71 87 14

Sephacryl 200-HR^d 30 34 >99 22 40 57 92 7 50 58 91 13 ^a The purity of isomer 1 is calculated from the total amount of protein combined with densitometric analysis of non-reducing Coomassie stained SDS-polyacrylamide gels. ^bThe Q Sepharose Fast Flow media was heavily overloaded with protein resulting in a significant loss of rAll in the flow through.

SDS-PAGE analysis of inclusion body-derived MHC-I heavy chains

Reducing SDS-PAGE analysis of solubilised inclusion body preparations revealed a monomeric band of high intensity with an apparent molecular mass of 32 kDa, which corresponds well with the theoretical molecular masses of the truncated recombinant heavy chain monomers. However, non-reducing SDS-PAGE analysis revealed a number of monomeric heavy chain isomers, which could only differ from the reduced isomer in their disulfide bond configuration (Fig. 4), in a similar manner to that which we have previously reported for rA2 (Pedersen et al., 2001). The band with the lowest mobility is the fully reduced heavy chain isomer (designated 0 in Fig. 4), whereas partially/fully oxidised species have a higher mobility (designated 1, 2 in Fig. 4). Only one of these oxidised forms have attained the correct disulfide bond configuration. In the case of rA2 and rAll only oxidized species were observed, whereas for rK^k (des cys) the reduced form was also found. Isomers 1 and 2 were present in equimolar amounts in the rA2 preparation, but in all other preparations isomer 1 was the dominant form. Thus, more than 90 % of the observed rK (des cys) heavy chain forms were accounted for by isomer 1, indicated that this isomer is the most stable one.

Separation of oxidized species of MHC-I heavy chain monomers In order to determine the most effective refolding candidate among the observed oxidised heavy chain isomers, we screened several chromatographic separation techniques (i.e. hydrophobic interaction, anion-, cation- exchange chromatography) and media for their ability to resolve the different isomers. Although the anion-exchanger Q Sepharose Fast Flow was able to partly separate isomers 1 and 2 in the rA2 preparation (results not shown), the separation was much better when using hydrophobic interaction chromatography (HIC). Several HIC media were tested, including, Butyl and Octyl Sepharose Fast Flow and three different versions of the Phenyl Sepharose media. In all cases however, the best resolution was achieved on Phenyl Sepharose High Performance. Figures 5 and 6 respectively show chromatograms corresponding to the fractionation of rA2 and rAll on this media and the non-reducing SDS-PAGE analysis of selected fractions. In the case of rAll, isomer 1 eluted first, whereas the order was reversed for rA2. An additional isomer, not discovered during the initial analysis, appeared during the fractionation of rAll and was designated isomer 3 (Fig. 6). The band representing isomer 3 is more diffuse than bands 1 and 2 and could conceivably represent more than one band, corresponding to different disulfide bond isomers of rAll. Isomer 3 co-eluted with the rAll isomer 1 and it was not possible to resolve the two with any of the chromatographic techniques tested in this study. High molecular weight contaminants and multimerised forms of the heavy chain monomer were removed by size exclusion chromatography (SEC) on Sephacryl 200-HR (rA2 & rAll) and Sephacryl 400-HR (rK^k (des cys)). By combining anion-exchange on Q Sepharose Fast Flow with HIC on Phenyl Sepharose High Performance and SEC the final purities of heavy chain preparations were about 95% as estimated by densitometric analysis of non- reducing SDS-polyacrylamide gels and yields were around 20-30 mg/l bacterial culture. Table 1 shows a summary of the purification steps for all three heavy chains.

Analysis of peptide binding to fractionated oxidised heavy chain isomers

The ability of fractionated heavy chains to undergo productive refolding in the presence of β₂m and a specific radiolabeled peptide at pH 6.6 was examined with a biochemical-binding assay (see Materials and methods). In short, samples were withdrawn from collected fractions containing heavy chain monomer and assayed for peptide binding. To avoid ligand depletion, which would lead to peptide binding affinity being underestimated, each sample was diluted in 8 M Urea to obtain a final heavy chain concentration in the range of 10-30 nM.

Figure 7 shows the peptide binding analysis performed on fractions collected during purification of rA2 and rAll on Phenyl Sepharose High Performance. From Figure 7 it is evident that the peptide-binding signal coincides with the protein tracing for isomer 1 and not for isomer 2 for both rA2 (Fig. 7A) and rAll (Fig. 7B). A similar profile although not shown was observed during purification of rK^k (des cys). The peptide binding observed in fractions enriched in isomer 2 is most likely the result of low amounts of isomer 1 present in these fractions (Fig. 6, 7B; compare the analysis of fraction 55 and 120).

Evaluation of refolding efficiency with a quantitative and conformationally sensitive ELISA

In the case of rAll, isomer 1 co-eluted with isomer 3, making it difficult to determine whether only one or both of them were contributing to the observed peptide-binding signal (Fig. 5). Based on the results presented in Figure 7, isomer 1 is likely to be refolding properly and binding peptide as one fraction enriched in this isomer, but essentially free of isomer 3, gave rise to a peptide-binding signal of 15% (Fig. 6, 7B; fraction 95).

To further pursue the question of refolding efficiency and peptide-binding abilities of rAll heavy chain isomers 1 and 3 a newly developed quantitative ELISA was exploited

(Sylvester-Hvid et al., 2002). When β₂m and peptide are present at saturating conditions, this ELISA assay allows the determination of the concentration of heavy chain molecules, which can fold into complexes.

The refolding reaction was performed with a purified batch of rAll, enriched in isomer 1, but also containing very low amounts of isomer 3. Figure 8 shows a non-reducing SDS- PAGE analysis of the tested rAll preparation as well as the processed results from the ELISA analysis. A linear fit to the ELISA data (equation: y = 0.96-x - 4.14, R² = 0.99) estimates the amount of heavy chain available for complex formation to about 96 % of the total amount. By comparison the densitometric analysis of the non-reducing SDS- polyacrylamide gel in Figure 8 gives the following distribution of isomers 1 and 3: 92 % and 8 %, respectively. This demonstrate that isomer 1 is the active form of the two isomers.

Comparison of refolding efficiency between the correctly oxidised (isomer 1) and fully reduced (isomer 0) rAll heavy chain monomer

To evaluate the performance of the chosen method, the refolding of the apparently correctly oxidised heavy chain monomer (isomer 1) was compared with that of the fully reduced monomer (isomer 0). An inclusion body preparation of rAll was solubilised under non-reducing conditions and purified on Phenyl Sepharose High Performance as described in Materials and methods. Fractions enriched in isomer 1 and completely free from isomer 2 were pooled and then divided into two equal aliquots. One of them was reduced by an overnight treatment with 4 mM DTT at 4°C.

Subsequently, both were purified on Sephacryl 400-HR to remove high molecular weight contaminants. For the reduced heavy chain preparation, the buffer was supplemented with 2 mM DTT to prevent re-oxidation during the size exclusion chromatography. The resulting heavy chain preparation only contained reduced species as ascertained by non-reducing SDS-PAGE mobility analysis (data not shown).

Fractions containing highly purified rAll monomer were pooled and the ability of the pooled material to undergo productive refolding at acidic pH in the presence of β₂m and a radiolabeled peptide was examined (Fig. 9). Binding of 10% of the offered peptide was reached at a concentration of only 30 nM of isomer 1, whereas 150 nM of reduced heavy chain was required to reach the same degree of binding, demonstrating that isomer 1 undergoes refolding with considerably higher efficiency than the fully reduced heavy chain (isomer 0). In summary, it has been shown that oxidised species of heavy chain monomers can be separated by hydrophobic interaction chromatography under non-reducing and denaturing conditions and that one of these isomers is able to undergo efficient refolding and simultaneously peptide binding under acidic conditions. The feasibility of the suggested production and purification process was demonstrated for both murine and human MHC-I molecules and it is believed that it can be extended to include all MHC-I molecules as well as a range of other proteins.

REFERENCES

Altman, J. D., Moss, P. A., Goulder, P. .., Barouch, D. H., McHeyzer-Williams, M. G., Bell, 3. I., McMichael, A. J. and Davis, M. M. (1986): Phenotypic analysis of antigen-specific T lymphocytes. Science. 274, 94-6.

Bernard, A. and Payton, M. (2002): Fermentation and Growth of Escherichia coli for optimal protein production. In Current Protecols of Protein Science, (Eds Coligan, J.E., Dunn, B.M., Speicher, D.W., Wingfield, P.T.), Unit 5.3, John Wiley & Sons.

Buus, S., Stryhn, A., Winther, K., Kirkby, N. and Pedersen, L. 0. (1995): Receptor-ligand interactions measured by an improved spun column chromatography technique. A high efficiency and high throughput size separation method. Biochim. Biophys. Acta. 1243, 453- 460

Buus, S. (1999): Description and prediction of peptide-MHC binding: the 'human MHC project'. Curr. Opin. Immunol. 11, 209-13

Chen, M. and Christen, P. (1997): Removal of chromosomal DNA by Mg2+ in the lysis buffer: an improved lysis protocol for preparing Escherichia coli whole-cell lysates for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal, Biochem. 246, 263-4.

Garboczi, D. N., Hung, D. T. and Wiley, D. C. (1992): HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc. Natl. Acad. Sci. USA. 89, 3429-33.

Laemmli, U. K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680-85

Maniatis et al., (1989) in Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) : Molecular cloning: a laboratory manual. 2nd edition, Cold Spring Harbour Press, USA, chapter 17

Rudolph, R. and Lilie, H. (1996) : In vitro folding of inclusion body proteins. Faseb ., 10, 49-56.

Springer, T.A., Robb, R ., Terhorst, C. and Strominger, J.L. (1979): Subunit and disulfide structure of monomeric and dimeric forms of detergent-soluble HLA antigens. J. Biol.Chem. 252, 4694-4700.

Sylvester, H. C, (2002): Measurering peptide-MHC class I affinity by a quantitative ELISA. Tissue Antigens, in press.

Claims

A method of purifying, from a population of different disulphide bonded isoforms of the same monomeric protein, the isoform(s) of optimal refolding ability, the method comprising the steps of

(i) solubilising said population by adding a denaturant,

(ii) subjecting said solubilised population to at least one separation step, under denaturing and non-reducing conditions, resulting in the separation of at least one isoform,

(iii) obtaining the isoform(s) of optimal refolding ability, and

(iv) subjecting the purified isoform(s) to a refolding treatment.

2. A method according to claim 1, wherein the solubilised proteins in step (i) are denatured under non-reducing conditions.

3. A method according to claim 1, wherein the solubilised proteins in step (i) are denatured under reducing conditions.

4. A method according to claim 3, wherein the reducing agents is selected from the group consisting of dithiothreitol, dithioerytritol, gluthathione, cysteine, cystamine and 2- mercaptoethanol.

5. A method according to claim 3, wherein the denatured and reduced proteins are oxidised before being subjected to the separation in step (ii).

6. A method according to claims 5, wherein the redox potential during oxidisation is adjusted using a mixture of reductant and oxidant.

7. A method according to any of claims 1-6, wherein the redox potential during the renaturation treatment is adjusted using a mixture of reductant and oxidant.

8. A method according to claim 6 or 7, wherein the redox pair is selected from the group consisting of reduced dithiothreitol/oxidised dithiothreitol; reduced glutathione (GSH)/oxidized glutathione (GSSG); cystamine/cysteamine.

9. A method according to any of claims 1-8, wherein the protein is produced as an aggregate.

5 10. A method according to any of claims 1-9, wherein the protein is produced as bacterial inclusion bodies.

11. A method according to any of claims 1-10, wherein the protein contains at least two cysteine residues.

10

12. A method according to any of claims 1-11, wherein the protein is an immunoglobulin superfamily protein.

13. A method according to claim 12, wherein the protein is an immunoglobulin superfamily 15 protein 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 histocompatibility molecules (MHC), β₂microglobulin (β₂m), lymphocyte function associated antigen-3 (LFA-3) and FcγRIII, CD7, CD8, Thy-1 and Tp44 (CD28), T cell receptor, CD4, polyimmunoglobulin 20 receptor, neuronal cell adhesion molecule (NCAM), myelin associated glycoprotein (MAG), P myelin protein, carcinoembryonic antigen (CEA), platelet derived growth factor receptor (PDGFR), colony stimulating factor-1 receptor, αβ-glycoprotein, ICAM (intercellular adhesion molecule), platelet and interleukins.

25 14. A method according to claim 13, wherein the immunoglobulin superfamily protein is a MHC.

15. A method according to claim 14, wherein the MHC protein is a murine or a human MHC.

30

16. A method according to claim 14 or 15, wherein the MHC protein is a MHC class I protein selected from the group consisting of a heavy chain, a heavy chain combined with a β₂m, and a functional mature MHC class I protein; or a MHC class II protein selected from the group consisting of an α/β dimer and an α/β dimer with a peptide.

35

17. A method according to any of claims 14-16, wherein the produced MHC protein is obtained as a peptide free MHC protein.

18. A method according to any of claims 1-17, wherein the produced protein is a substantially pure MHC monomer heavy chain.

19. A method according to any of claims 1-20, wherein the refolding treatment is carried 5 out at a pH value that are not necessarily optimal for disulphide bond formation.

20. A method according to claim 19, wherein the refolding treatment is carried out a neutral or acidic pH, i.e. less than or equal to pH 7.

10 21. A method according to claim 20, wherein the refolding treatment is carried out at pH 6.6

22. A method according to any of claims 1-21, wherein an agent which inhibits proteolysis is added during the refolding treatment.

15

23. A method according to claim 22, wherein the proteolysis inhibitor(s) is selected from the group consisting of N-ethyl-maleimide, pepstatin, phenyl methyl sulphonic flouride (PMSF) and EDTA, respectively, and of ATP dependent proteolysis inhibitors such as sodium ortho vanadate.

20

24. A method according to any of claims 1-23, wherein the proteins are solubilised under denaturing conditions using a denaturant selected from the group consisting of organic solvents, chaotrophic agents, detergents and/or salts.

25 25. A method according to claim 24, wherein the denaturant is urea at a concentration in the range of 0,5 - 9 M such as about 8 M.

26. A method according to any of claims 1-25, wherein the refolding treatment is performed in the presence of a reducing agent at a concentration which will affect

30 reduction of inappropriate disulphide bonds without affecting formation of appropriate bonds.

27. A method according to any of claims 1-26, wherein the refolding treatment is performed essentially in the absence of reducing agents.

35

28. A method according to any of claims 1-27, wherein the isoform(s) of optimal refolding ability is identified using an electrophoretic technique.

29. A method according to claim 28, wherein the electrophoretic technique is a non- reducing SDS-PAGE.

30. A method according to any of claims 1-29, wherein the isoform(s) of optimal refolding 5 ability is separated using a technique selected from the group consisting of dialysis, filtration, dia-filtration, tangential flow-filtration, gel-filtration, extraction (two-phase extraction), precipitation, centrifugation, electrophoretic techniques and chromatographic techniques.

10 31. A method according to any of claims 1-30, wherein the isoform(s) of optimal refolding ability is purified using a combination of ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography.

32. A method according to any of the preceding claims, wherein the produced protein has 15 a purity of at least 70%.

33. A functionally active MHC heavy chain protein obtainable by a method according to any of claims 1-32.

20 34. Use of a MHC protein according to claim 33 in analysis of peptide binding capacity.

25