EP1183364A1 - Groel muteins with improved stability - Google Patents

Abstract

A GroEL chaperone polypeptide, or homologue thereof, or fragment thereof having protein refolding activity, comprising one or more amino acid modifications at any one of amino acid residues (207, 212, 217, 223, 233, 267, 271, 294, 305, 308 and 326) of the GroEL amino acid sequence shown as SEQ I.D. No. 1 or their equivalent positions in other homologous chaperone polypeptides is provided.

Description

GROEL MUTEINS WITH IMPROVED STABILITY

Field of the invention

The present invention relates to chaperone polypeptides having improved stability which are active in the folding and maintenance of structural integrity of other proteins.

Background to the Invention

Chaperones are in general known to be large multisubunit protein assemblies essential in mediating polypeptide chain folding in a variety of cellular compartments. Several families of chaperones have been identified, for example the chaperonin hsp60 family, otherwise known as the cpn60 class of proteins, which are expressed constitutively. Members of the chaperonin hsp60 family have been identified in the bacterial cytoplasm (GroEL), in endosymbiotically derived mitochondria (hsp60) and in chloroplasts (Rubisco binding protein). Another chaperone family, designated TF55/TCP1 , is found in the thermophilic archaea and the evolutionarily connected eukaryotic cytosol. A comparison of amino acid sequence data has shown that there is at least 50% sequence identity between chaperones found in prokaryotes. mitochondria and chloroplasts.

The molecular chaperone GroEL assists the folding of many newly synthesized proteins in Escherichia coli (E. coli) (Gething & Sambrook. 1992). The active site of GroEL, which is responsible for the binding of polypeptide substrates, is located on two helices H8 and H9 as well as an adjacent loop (residues 199 to 204) of the apical domain (residues 191 to 376) (Fenton et al. , 1994; Buckle et al, 1997). Polypeptides corresponding to the apical domain of GroEL and fragments thereof, termed minichaperones, are active in vitro, facilitating the refolding of rhodanese and cyclophilin A (CyPA) and catalysing the unfolding of native barnase (Zahn et al, 1996), and in vivo complementing temperature sensitive mutants of GroEL (Chatellier et al., 1998). Thus, the essential chaperone activity does not require the allosteric properties and central cavity provided by intact GroEL, which are refinements for optimal activity (Ben-Zvi et al, 1998). When monodispersed on a solid support, minichaperones are highly active in assisting the renaturation of other proteins (Altamirano et al . 1997). "Refolding chromatography" based on this technique has immense potential in the biotechnology and pharmaceutical industries, in particular, when combined with immobilized protein disulphide isomerases (Altamirano et al.. 1999).

However, protein refolding in vitro often takes place over a range of conditions that are not suited to the use of wild type minichaperone polypeptides, such as high urea or guanidium chloride concentrations or high temperatures, since the wild type minichaperone polypeptides are not stable under such conditions. Indeed, protein stability is not generally optimized during evolution. For example, the stability of the minichaperone GroEL(193- 345) is relatively low (L G»J of 6.6 kcal mob¹).

Thus there is a need for improved minichaperone polypeptides having enhanced stability. A more stable minichaperone could be used over a wide range of more stringent conditions, such as high denaturant concentration or high temperature. Although an enhancement in protein stability may be achieved in theory by designed amino acid substitutions, a rational approach to stabilization is extremely difficult since it is not generally possible to predict the energetic and structural response to mutation. The statistics of isolated helices and parts of sheets are predictable to various extents (Regan et al. 1996). but effects in proteins are often strongly dependent on the structural context.

One possible approach, which takes advantage of naturally occurring variations among a family of homologous proteins, has provided several successful examples (Serrano et al. , 1993; Steipe et al, 1994; Shih & Kirsch, 1995). The strategy is essentially as follows. First the sequences of family members are aligned. Residues at positions of low conservation within the family are replaced by the representative residues as suggested by the alignment. The stability of the resulting set of single mutants is then measured. Stabilizing mutations are then combined with the aim of creating a "'super-stable" variant, since the stabilizing effects of mutations are approximately additive when the residues concerned are not in contact. This procedure has been used to stabilize the tumor suppressor p53 DNA binding domain (Nikolova et al., 1998). Summary of the invention

As part of an overall effort to widen and enhance the activity of minichaperones we have undertaken a mutagenesis study with the aim of creating a more stable protein. There are 130 sequences of homologous Cpn60 proteins available in the sequence databank. Sequence alignment of their apical domains shows that whereas the overall sequence homology is rather good, the conservation at particular positions is relatively low. The minichaperone corresponding to the apical domain of GroEL is thus ideally suited to a homology based mutagenesis study. Using the sequence alignments, we have substituted amino acid residues in the GroEL sequence with the aim of obtaining more stable variants. We have identified at least thirteen substitutions that increase protein stability. By combining the ^■■.-,.- mlizing mutations we have created two multiple mutants that have significantly increased stability while retaining full chaperone activity.

Accordingly the present invention provides a GroEL chaperone polypeptide. or homologue thereof, or fragment thereof having protein refolding activity, comprising one or more amino acid modifications at any one of amino acid residues 207, 212, 217. 223, 233, 267, 271. 294. 305, 308 and 326 of the GroEL amino acid sequence shown as SEQ I.D. No. 1 or their equivalent positions in other homologous chaperone polypeptides.

Preferably the modifications are amino acid substitutions. Preferably said substitutions are selected from Lys207→Asn. Ala212→Glu, Ser217→Asp, Ala223→Thr, Ala223→Val. Met233→Leu, Met267→Leu. Val271→Leu. Val271→Ser, Thr294→Arg, Ile305→Leu and Glu308— »Lys. Asn326— Thr, and their equivalents in other homologous chaperone polypeptides.

More preferably the polypeptide of the invention comprises a combination of at least two amino acid modifications. A particularly preferred combination is [Ala212-->Glu, Ala223→Thr, Met233→Leu, Ile305→Leu, Glu308→Lys and Asn326→Thr] or [Ala212→Glu, Ala223→Val, Met233→Leu, Ile305→Leu, Glu308→Lys and Asn326— Thr], or their equivalents in other homologous chaperone polypeptides. Preferably. the polypeptide of the invention consists essentially of amino acids 193 to 335, 191 to 337, 191 to 345 or 191 to 376 of GroEL or the equivalent residues of homologous chaperone polypeptides.

Preferably, the polypeptide of the invention, when in solution, remains monomeric and has the ability to refold, reactivate or recondition proteins.

The present invention also provides a nucleic acid molecule encoding a polypeptide of the invention. Also provided is a vector comprising a nucleic acid of the invention, optionally operably linked to a regulatory sequence capable of directing expression of said nucleic acid in a suitable host cell. A host cell comprising a nucleic acid or a vector of the invention is also provided.

In another aspect the present invention provides a method of making a polypeptide of the invention comprising transforming a host cell with a nucleic acid encoding said polypeptide, culturing the transformed cell and expressing said polypeptide.

The present invention further provides a pharmaceutical composition comprising a polypeptide of the invention or a nucleic acid of the invcNon together with a pharmaceutically acceptable diluent, carrier or excipient.

In a further aspect, the present invention provides a polypeptide. nucleic acid, vector or pharmaceutical composition of the invention for use in therapy. Also provided is the use of a polypeptide, nucleic acid, vector or pharmaceutical composition of the invention for use in the manufacture of a medicament for the treatment of disease associated with protein/polypeptide structure.

The present invention also provides a method of reconditioning a molecule comprising contacting said molecule with the chaperone polypeptide of the invention. Preferably the molecule is a protein/polypeptide. Preferably, the molecule is subjected to inactivation or denaturation prior to contacting with said chaperone polypeptide. Preferably the chaperone polypeptide is immobilised to a solid phase. More preferably the solid phase is a chromatographic matrix and the contacting of the molecule and chaperone polypeptide is carried out by applying the molecule to the top of a bed of the matrix packed in a column and then eluting the molecule through the column.

The present invention further provides the use of a polypeptide of the invention for altering the structure of a molecule. Preferably the molecule is a protein or polypeptide and the alteration in structure is by folding, unfolding or refolding.

In a preferred embodiment, the stoichiometry between the chaperone polypeptide and the molecule being altered is about 1 : 1.

The present invention also provides the use of a polypeptide of the invention for purifying or increasing the yield, specific activity or quality of biological molecules.

Also provides is a kit for reconditioning or refolding a molecule comprising a polypeptide according to the invention immobilised to a solid phase and a container for holding said solid phase polypeptide.

In another aspect the invention provides the use of a chaperone polypeptide of the invention in the production of a protein or polypeptide by recombinant means, wherein the said chaperone polypeptide is co-expressed with the protein or polypeptide thereby to improve the yield, specific activity or quality of the protein or polypeptide.

In a further aspect the invention provides a method of treating a human or animal patient suffering from a disease associated with protein/polypeptide structure which method comprises administering to a patient an effective amount of a polypeptide or polynucleotide of the invention Detailed description of the invention

Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al.. Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.

A. Pol^ypeptides

GroEL is a member of the hspόO family of heat shock proteins. GroEL is a tetradecamer wherein each monomeric subunit (cpnόOm) has a molecular weight of approximately 57 kDa. The tetradecamer facilitates the in vitro folding of a number of proteins which would otherwise misfold or aggregate and precipitate. The structure of GroEL from E- coli has been established through X-ray crystallographic studies (see Braig K. et al, 1994. The holo protein is cylindrical, consisting of two seven-membered rings that form a large central cavity which is generally considered to be essential for activity. Some small proteins have been demonstrated to fold from their denatured states when bound to GroΕL and it has been argued that a cage-like structure is necessary to sequester partly folded or assembled proteins.

The entire amino acid sequence of E. coli GroΕL is also known (see Braig K et al, 1994) and three domains have been ascribed to each cpnόOm of the holo chaperonin (tetradecamer). These are the intermediate (amino acid residues 1-5, 134-190, 377-408 and 524-548), equatorial (residues 6-133 and 409-523) and apical (residues 191 -376) domains.

GroΕL facilitates the folding of a number of proteins by two mechanisms; (1) it prevents aggregation by binding to partly folded proteins, which then refold on GroΕL to a nativelike state and (2) it continuously anneals misfolded proteins by unfolding them to a state from which refolding can start again.

There are four key properties that may characterise a protein as a molecular chaperone (1) suppression of aggregation during protein folding; (2) suppression of aggregation during protein unfolding; (3) influence on the yield and kinetics of folding; and (4) effects exerted at near stoichiometric levels.

Chaperone activity may be determined in practice by an ability to refold cyclophilin A but other suitable proteins such as glucosamine-6-phosphate deaminase or a mutant form of indoleglycerol phosphate synthase (IGPS) (amino acid residues 49-252) may be used. A rhodanese refolding assay may also be used. Details of a suitable refolding assay are given below and in the examples.

Preferred chaperone polypeptides of the present invention have protein refolding activity in the absence of adenosine triphosphate of more than 50%, preferably 60%. even more preferably 75%, ----- J refolding activity being determined by contacting the chaperone polypeptide with an inactivated protein of known specific activity prior to inactivation, and then determining the specific activity of the said protein after contact with the polypeptide. the % refolding activity being:

specific activity of protein after contact with polypeptide x 100 specific activity of protein prior to inactivation 1

Preferably, the chaperone activity is determined by the refolding of cyclophilin A. More preferably. 8 M urea denatured cyclophilin A (100 μM) is diluted into 100 mM potassium phosphate buffer pH7.0, 10 mM DTT to a final concentration of l μM and then contacted with at least lμM of said polypeptide at 25°C for at least 5 min, the resultant cyclophilin A activity being assayed by the method of Fischer et al. (1984).

It is preferred that chaperone polypeptides of the present invention are monomeric in solution and incapable of multimerisation in solution. Monomeric GroEL minichaperones are disclosed in W098/13496. Typically, multimerisation is prevented by using chaperone polypeptides that lack the interacting domains found outside the apical domain, although it could be achieved by suitable mutations.

A GroEL chaperone polypeptide, or homologue thereof, of the present invention comprises at least one modified amino acid residue selected from amino acids 207, 212, 217, 223, 233, 267, 271. 294, 305, 308 and 326 of the GroEL amino acid sequence shown as SEQ ID No. 1 or their equivalent positions in other homologous chaperone polypeptides. It is preferred that said modifications are amino acid substitutions. In particular is it preferred that the amino acid substitutions are selected from the following specific substitutions exemplified herein, i.e. Lys207— Asn, Ala212-»Glu, Ser217— ->Asp, Ala223— - Thr, Ala223→Val. Met233→Leu, Met267→Leu, Val271→Leu. Val271→Ser. Thr294→Arg, Ile305— Leu and Glu308-»Lys, Asn326— - Thr. and their equivalents in other homologous chaperone polypeptides.

It is especially preferred to use combinations of modifications to further enhance protein stability, such as two or more modifications. In a particularly preferred embodiment, the polypeptide of the invention comprises modifications selected from [Ala212— - Glu, Ala223→Thr, Met233→Leu, Ile305->Leu. Glu308→Lys and Asn326→Thr], and [Ala212→Glu, Ala223→Val, Met233→Leu, Ile305→Leu, Glu308→Lys and Asn326— »Thr], and their equivalents in other homologous chaperone polypeptides.

With respect to fragments, polypeptides of the invention will generally comprise at least amino acids 193 to 335 of GroEL. or their equivalents in other chaperone polypeptides. Other preferred fragments include amino acids 193 to 335, 191 ie 7, 191 to 345 and 191 to 376 of GroEL or their equivalents in other chaperone polypeptides.

It will be understood that the modified polypeptides of the invention are not limited to GroEL polypeptides based on the amino acid sequence set out in SEQ. ID. No. 1 or fragments thereof having chaperone activity but also include homologous chaperone polypeptides sequences obtained from any source, for example related viral/bacterial proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof.

Thus polypeptides of the invention may also be based on chaperone homologues from other species including bacteria such as E. coli, yeast and animals such as mammals (e.g. mice, rats or rabbits), especially primates, more especially humans. Thus. the present invention covers variants, homologues or derivatives of the amino acid sequence set out in SEQ ID No. 1 which comprise similar stabilising modifications as those described for GroEL, as well as variants, homologues or derivatives of the nucleotide sequences coding for the amino acid sequences of the present invention.

It is preferred that modified chaperone polypeptides of the invention have an increased stability, as measured by ΔΔGι_D»»ι--. , of at least - 0.1 kcal mob¹, preferably at least -0.2, -0.4,

-0.5 or -1.0 kcal mob¹ relative to the unmodified sequence, more preferably by at least -2.0 or -5.0 kcal mob' . ΔG;«-ι» may be determined as described in the examples.

In the context of the present invention, a homologous sequence is taken to include an amino acid sequence which is at least 30, 40, 50. 60. 70. 80 or 90% identical, preferably at least 95 or 98% identical at the amino acid level over at least 50 or 100. preferably 200, 300. 400 or 500 amino acids with SEQ ID No. 2. In particular, homology should typically be considered with respect to those regions of the sequence known to be essential for protein function rather than non-essential neighbouring sequences. For example, homology may be compared with amino acids 191 to 376 (apical domain) of SEQ ID No. 1.

Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye. or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.

% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids). Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs^" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al, 1984. Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to. the BLAST package (see http://www.ncbi.nih.gov/BLAST/). FASTA (Atschul et al, 1990, J. Mol. Biol., 403-410; FASTA is available for online searching at, for example, http://www.2.ebi.ac.uk.fasta3) and the GENEWORKS suite of comparison tools. However it is preferred to use the GCG Bestfit program. Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate % homology, prefe---u y % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

The skilled person can identify suitable homologues by, for example, carrying out a search of online databases using all or part of SEQ ID. No. 1 as a query sequence. For example, a search of the Swissprot database using the BlastP program Ver 2.0.8 (default settings) (Jinghui Zhang et al, 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs". Nucleic Acids Res. 25:3389-3402) and ammo acids 191 to 376 of SEQ ID No. 1 as the query sequence identified well over a hundred homologous sequences, many of which gave homology scores of at least 50% identity. Homologues identified include members of the hspόO chaperonin family which includes the eubacterial GroEL, mitochondrial hspόO and chloroplast cpn60. Other specific homologues together with their database accession numbers are detailed in W098/13496.

The terms "variant" or "derivative" in relation to the amino acid sequences of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence retains substantially the same activity as the unmodified sequence, preferably having at least the same activity as the wild type polypeptide from which the variant or derivative is derived (such as the GroEL sequence shown in SEQ ID. No. 1). A suitable assay for determining activity is described above. Polypeptides having the amino acid sequence shown in SEQ I.D. No. 1 , or fragments or homologues thereof may be modified for use in the present invention. Typically, modifications are made that maintain the biological activity of the sequence. Amino acid substitutions may be made, for example from 1 , 2 or 3 to 10. 20 or 30 substitutions provided that the modified sequence retains the biological activity of the unmodified sequence. Guidance is given herein as to certain modifications that enhance stability of the chaperone polypeptides of the present invention. Guidance is also given herein as to modifications that do not impair protein stability as well as modifications that reduce protein stability. Modifications that reduce protein stability should be avoided. Amino acid substitutions may include the use of non-naturally occurring analogues, for example to increase blood plasma half-life of a therapeutically administered polypeptide.

Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

Polypeptides of the invention also include fragments of the full length sequences mentioned above having chaperone activity. Preferably said fragments comprise at least the apical domain of GroEL or its equivalent in chaperone homologues. Polypeptides of the invention may further comprise heterologous amino acid sequences, typically at the N-terminus or C-terminus. preferably the N-terminus Heterologous sequence may include sequences that affect intra or extracellular protein targeting (such as leader sequences). Heterologous sequences may also include sequences that increase the immunogenicity of the polypeptide of the invention and/or which facilitate identification, extraction and/or purification of the polypeptides. Another heterologous sequence that is particularly preferred is a polyamino acid sequence such as polyhistidine which is preferably N-terminal. A polyhistidine sequence of at least 10 amino acids, preferably at least 17 amino acids but fewer than 50 amino acids is especially preferred.

Polypeptides of the invention are typically made by recombinant means, for example as described below. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Polypeptides of the invention may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), όxHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences, such as a thrombin cleavage site. Preferably the fusion protein will not hinder the function of the protein of interest sequence.

The use of appropriate host cells is expected to provide for such post-translational modifications (e.g. myristolation, glycosylation. truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Thus, for example, mammalian homologues may preferably be expressed using mammalian host cells.

Polypeptides of the invention may be in a substantially isolated form. It will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99%) of the protein in the preparation is a polypeptide of the invention.

A polypeptide of the invention may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, e.g. ^I23I, enzymes, antibodies, polvnucleotides and linkers such as biotin.

A polypeptide or labelled polypeptide of the invention or fragment thereof may also be fixed to a solid phase, for example a chromatographic matrix such as sepharose. Such labelled and/or immobilised polypeptides may be packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.

B. Polvnucleotides

Polvnucleotides of the invention comprise polvnucleotides encoding the polypeptides of the invention. It will be understood by a skilled person that numerous different polvnucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may. using routine techniques, make nucleotide substimtions that do not affect the polypeptide sequence encoded by the polvnucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

Polvnucleotides of the invention may comprise DNA or RNA. They may be single- stranded or double-stranded. They may also be polvnucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life ^• span of polynucleotides of the invention. l

The terms "variant", "homologue" or "derivative" in relation to the nucleotide sequence of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence. Preferably said variant, homologues or derivatives code for a polypeptide having biological activity, preferably having substantially the same activity as the GroEL apical domain (for example, amino acids 191 to 376 of the amino acid sequence shown as SEQ ID No. 1 ).

Polynucleotides for use in the present invention, for example as a starting point for the construction of modified polypeptides of the invention will tvpically have at least 50 or 75% sequence homology. more preferably at least 85%. more preferably at least 90%) or 95% homology to the - - quence shown SEQ ID. No. 2 over a region of at least 20, preferably at least 25 or 30. for instance at least 40, 60 or 100 or more contiguous nucleotides. Preferred polynucleotides for use in the invention will comprise regions encoding polypeptide domains homologous to the apical GroEL domain, preferably at least 70, 80 or 90% and more preferably at least 95% homologous to said regions.

Nucleotide homology comparisons may be conducted as described above for polypeptides. A preferred sequence comparison program is the GCG Winsconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and - 9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.

Such nucleotide sequences for use in the present invention are typically capable of hybridising selectively to SEQ ID. No. 2 or any variant, fragment or derivative thereof, or to the complement of any of the above. Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.

The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction technologies. The term "selectively hybridizable" means that the polynucleotide used as a probe is used under conditions where a target polynucleotide of the invention is found to hybridize to the probe at a level significantly above background. The background hybridization may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screening. In this event, background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with ^J~P.

Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques. Methods in Enzymology. Vol 152, Academic Press. San Diego CA), and confer a defined "stringency" as explained below.

Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm: intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybπ^'dization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.

Where the polynucleotide of the invention is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the present invention. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included within the scope of the present invention.

Polynucleotides of the invention or for use in the present invention which are not 100% homologous to SEQ ID. No. 2 can be obtained in a number of ways. For example, other viral/bacterial, or eukaryotic homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other species, and probing such libraries with probes comprising all or part of SEQ I.D. No 2 under conditions of medium to high stringency.

Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences, such as SEQ ID. No 2. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

Polynucleotides of the invention or for use in the present invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length.

Polynucleotides such as a DNA polynucleotides and probes may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time Techniques for accomplishing this using automated techniques are readily available in the art

Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques This will involve making a pair of primers (e g of about 15 to 30 nucleotides) flanking a region of the hpid targeting sequence which it is desired to clone, bnnging the pnmers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e g by purifying the reaction mixture on an agarose gel) and recovenng the amplified DNA The pnmers may be designed to contain suitable restnction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector

Polynucleotides or pnmers of the invention may carry a revealing label Suitable labels include radioisotopes such as ^J"P or ³S, enzyme labels, or other protein labels such as biotin Such labels may be added to polynucleotides or primers of the invention and may be detected using by techniques known pet se

C Nucleic acid vectors

Polvnucleotides ot the invention can be incorporated into a recombinant rephcable vector The vector may be used to replicate the nucleic acid in a compatible host cell Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a rephcable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector The vector may be recovered from the host cell Suitable host cells include bacteria such as E coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells Preferably. a polynucleotide of the invention in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.

Vectors of the invention may be transformed or transfected into a suitable host cell as described below to provide for expression of a protein of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein. Vectors will be chosen that are compatible with the host cell used.

The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used, for example, to transfect or transform a host cell.

Control sequences operably linked to sequences encoding the polypeptide of the invention include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in. The term promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers. The promoter is may typically be selected from promoters which are functional in prokaryotic cells or eukaryotic cells depending on the host cells in which it is desired to express the polypeptides of the invention. The promoter may be derived from promoter sequences of bacterial, viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of α-actin, β-actin. tubulin) or. alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.

It may also be advantageous for the promoters to be inducible so that the levels of expression of the polynucleotide of the invention can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.

In addition, any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.

D. Host cells

Vectors and polynucleotides of the invention may be introduced into host cells for the purpose of replicating the vectors/polynucleotides and/or expressing the polypeptides of the invention encoded by the polynucleotides of the invention. Suitable host cells include prokaryotes such as eubacteria, for example E. coli and B. subtilis and eukaryotes such as yeast, insect or mammalian cells. Vectors/polynucleotides of the invention may be introduced into suitable host cells using a \ anety of techniques known in the art, such as transfection, transformation and electroporation W^rhere vectors/polynucleotides of the invention are to be administered to animals, eral techniques are known in the art, for example infection with recombinant \ iral vectors such as retroviruses. herpes simplex viruses and adenoviruses, direct injection of nucleic acids and biohstic transformation

E_ Protein Expression and Purification

Host cells comprising polvnucleotides of the invention may be used to express polypeptides of the invention Host cells may be cultured under suitable conditions which allow expression of the proteins of the invention Expression of the polypeptides of the invention may be constitutive such that they are continually produced, or inducible, lequiπng a stimulus to initiate expression In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.

Polypeptides of the invention can be extracted from host cells by a variety of techniques known in the art. including enzymatic, chemical and/or osmotic lysis and physical disruption

Purification of polypeptides may optionally be performed using well known techniques such as affinity chromatography, including lmmunoaffimty chromatography ion-exchange chromatography and the like A particularly preferred technique is to express the polypeptide of the invention as a fusion protein with polyhistidine tag (for example 6xHιs) and purify cell extracts using Ni-NTA agarose (Qiagen) A variety of other similar affinity chromatography systems based on fusion protein sequences are known in the art

Polypeptides of the invention may also be produced recombinantly in an in vitro cell-free system, such as the TnT™ (Pro mega) rabbit reticulocyte system F. Antibodies.

The invention also provides monoclonal or polyclonal antibodies to polypeptides of the invention or fragments thereof. Thus, the present invention further provides a process for the production of monoclonal or polyclonal antibodies to polypeptides of the invention.

If polyclonal antibodies are desired, a selected mammal (e.g.. mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide bearing GroEL epitope(s). Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an orbit epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptemsed to another polypeptide for use as immunogens in animals or humans.

Monoclonal antibodies directed against epitopes in the polypeptides of the invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties: i.e., for isotype and epitope affinity.

An alternative technique involves screening phage display libraries where, for example the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs). This technique is well known in the art.

Antibodies, both monoclonal and polyclonal, which are directed against orbit epitopes are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy. Monoclonal antibodies, in particular, may be used to raise anti-idiotype antibodies. Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the agent against which protection is desired.

Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful in therapy.

For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab') and F(ab')₂ fragments, as well as single chain antibodies (scFv). Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.

G. Uses

Polypeptide chaperones of the invention may be used for altering the structure of a molecule, preferably a protein. Structural alterations include folding, unfolding and refolding. The effect of the alterations is preferably to improve the yield, specific activity and/or quality of the molecule. This may typically be achieved by resolubilising. reconditioning and/or reactivating incorrectly folded molecules post-synthesis and/or by acting on said molecules during their synthesis so as to ensure that the molecules adopt the correct conformation ab initio.

The terms "reconditioning" and "reactivating^"' are thus intended primarily to encompass in vitro procedures. Particular examples of in vitro procedures may include processing polypeptides that have been solubilised from cell extracts (such as inclusion bodies) using strong denaturants such as urea or guanidium chloride.

The terms "refold", "reactivate" and "recondition" are not intended as being mutually exclusive. For example, an inactive protein, perhaps denatured using urea, may have an unfolded structure. This inactive protein may then be refolded with a polypeptide of the invention thereby reactivating it. In some circumstances there may be an increase in the specific activity of the refolded/reactivated protein compared to the protein prior to inactivation/denaturation: this is termed "reconditioning".

The molecule is typically an unfolded or misfolded polypeptide which is in need of folding. Alternatively, however, it may be a folded polypeptide which is to be maintained in a folded state. Preferably, the polypeptide contains at least one disulphide linkage (or two cysteine residues capable of forming such as linkage under suitable conditions).

The invention envisages at least two situations. A first situation is one in which the polypeptide to be folded is in an unfolded or misfolded state, or both. In this case, its correct folding is promoted by the method of the invention. A second situation is one in which the polypeptide is substantially already in its correctly folded state, that is all or most of it is folded correctly or nearly correctly. In this case, the method of the invention serves to maintain the folded state of the polypeptide by affecting the folded/unfolded equilibrium so as to favour the folded state. This prevents loss of activity of an already substantially correctly folded polypeptide. These, and other, eventualities are covered by the reference to "promoting" the folding of the polypeptide.

As used herein, a polypeptide may be unfolded when at least part on? has not yet acquired its correct or desired secondary or tertiary structure. A polypeptide is misfolded when it has acquired an at least partially incorrect or undesired secondary or tertiary structure.

In vitro procedures will generally comprise contacting the molecule and the chaperone polypeptide of the present invention. The molecule may have been intentionally inactivated or denatured using, for example urea, prior to contacting with the chaperone polypeptide.

In a preferred aspect, the contact occurs with the molecular chaperone immobilised on a solid support. Examples of commonly used solid supports include beads, "chips", resins, matrices, gels, and the material forming the walls of a vessel. Matrices, and in particular gels, such as agarose gels, may conveniently be packed into columns. A particular advantage of solid phase immobilisation is that the reagents may be removed from contact with the polypeptide(s) with facility.

Immobilisation of the chaperone polypeptides of the present invention may be by covalent bonding or other means. In a preferred aspect of the present invention an immobilisation method is used which comprises a reversible thiol blocking step. This is important where the peptide contains a disulphide bond. Preferably, before protection the disulphides are reduced using a reducing agent such as DTT (dithiothreitol), under for example an inert gas. such as argon, to prevent reoxidation. Subsequently, the polypeptide is cyanylated, for example using NCTB (2-nitro, 5-thiocyanobenzoic acid) preferably in stoichiometric amounts, and subjected to controlled hydrolysis at high (non-acidic) pH. for example using NaHC0₃. The thiols are thus reversibly protected.

The chaperone polypeptide is then brought into contact with the solid phase component, for example at between 2.0 and 20.0 mg polypeptide/ml of solid component, preferably between 5.0 and 10.0 and most preferably around about 6.5 mg. The coupling is again carried out at a high (non-acidic) pH, for example using an NaHCOs coupling buffer.

Preferably, after coupling the remaining active groups may be blocked, such as with ethanolamine. and the uncoupled polypeptide removed by washing. Thiol groups may finally be regenerated on the coupled polypeptide by removal of the cyano groups, for example by treatment with DTE or DTT.

Treatment of the molecule which is to reconditioned, refolding, resolubilised and/or reactivated may be performed simply be incubating the molecule in aqueous solution with the chaperone polypeptide. The polypeptide may be immobilised to a solid phase as described above, preferably a chromatographic matrix, and the contacting of the molecule and chaperone polypeptide is carried out by applying the molecule to the top of a bed of the matrix packed in a column and then eluting the molecule through the column using standard techniques. It is particularly preferred that incubations take place under reducing conditions, which may, for example, be achieved using a combination of oxidised glutathione (GSSG) and glutathione (GSH) which act as a redox buffer system and prevent formation of disulphide bonds present in the oxidised state.

Preferably, the stoichiometry between the chaperone polypeptide of the invention and the molecule being altered is about 1 : 1. preferably 1 : 1.

The polypeptides of the invention may also be used in vivo. For example, a known problem associated with high level expression of polypeptides is that the large amounts of synthesised protein present in the cell cannot be processed properly by the cell's own machinery. In, for example, bacteria, this leads to deposits of insoluble protein in inclusion bodies that may only be solubilised in powerful denaturants such as urea and guanidium chloride. Co-expression of chaperone polypeptides of the invention may be used to assist the host cell in processing polypeptides expressed at high levels thus increasing the amount of properly folded soluble active protein which may be extracted using relatively mild techniques.

Thus the invention also provides for the use of a polypeptide of the invention in the production of a protein or polypeptide by recombinant means, wherein the said polypeptide is co-expressed with the protein or polypeptide thereby to improve the yield, activity and/or quality of the protein or polypeptide.

Chaperone polypeptides of the invention may be used in combination with another polypeptide that alters the structure of molecules, such as other components of cellular protein refolding machinery, for example foldases. Preferably, the foldase is selected from thiol/disulphide oxidoreductases and peptidyl prolyl isomerases. Specific examples include cyclophilin and thiol/disulphide oxidoreductases selected from E. coli DsbA and mammalian protein disulphide isomerase. Other polypeptides include members of the hsp70 family of chaperones, such as GroΕS. Further details of refolding methods using molecular chaperones together with foldases are described in WO99/05163, the contents of which are incorporated herein by reference. H. Therapeutic uses

Some human diseases, such as Alzheimer^'s disease and prion diseases including Creutzfeld-Jacob's disease are known to be associated with aberrant protein/polypeptide structure. Administration of polypeptides of the invention, either directly or via nucleic acid constructs, to affected cells may be used to the treatment of said diseases. The aberrant nature of the protein/polypeptide may be due to misfolding or unfolding which in turn may be due to an anomalous e.g. mutated amino acid sequence. The protein/polypeptide may be destabilised or deposited as plaques, for example as in Alzheimer's disease. The disease might be caused by a prion. A polypeptide-based medicament of tu-- invention would act to renature or resolubilise aberrant, defective or deposited proteins.

Consequently there is provided the use of a polypeptide of the invention for the manufacture of a medicament for the treatment of disease associated with aberrant protein/polypeptide structure. There is provided a nucleic acid molecule in accordance with other aspects of the invention for use in the treatment of disease. Consequently, there is provided the use of a nucleic acid molecule of the invention for the manufacture of a medicament for the treatment of disease associated with protein/polypeptide structure. Genetic therapy in vivo is therefore provided for by way of introduction and expression of DNA encoding the chaperone polypeptide in cells/tissues of an individual to provide chaperonin activity in those cells/tissues.

An antibody reactive against a polypeptide of the invention may also be used for the treatment of disease, such as disease associated with protein/polypeptide structure.

Polypeptides of the invention may also be used in a method of treating disease which comprises administering an effective amount of an inhibitor of the chaperone activity of a polypeptide of the invention. Preferably, said inhibitor is an antibody. L Administration

Polypeptides and/or antibodies of the invention may preferably be combined with various components to produce compositions of the invention. Preferably the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition of the invention may be administered by direct injection. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration. Typically, each protein may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.

Polynucleotides/vectors encoding polypeptide components for use in therapy may be administered directly as a naked nucleic acid construct. They may further comprise flanking sequences homologous to the host cell genome. When the polynucleotides/vectors are administered as a naked nucleic acid, the amount of nucleic acid administered may typically be in the range of from 1 μg to 10 mg, preferably from 100 μg to 1 mg. It is particularly preferred to use polynucleotides/vectors that target specifically tumour cells, for example by virtue of suitable regulatory constructs or by the use of targeted viral vectors.

Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents. Example of these agents include catio ic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam™ and transfectam™). Typically, nucleic acid constructs are mixed with the transfection agent to produce a composition.

Preferably the polynucleotide or vector according to the invention is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate- buffered saline. The composition may be formulated for parenteral. intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.

The routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient and condition.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. The Examples refer to the Figures. In the Figures:

Description of the Figures

Figure 1. Three-dimensional structure of minichaperone GroEL(191-345) (PDB ID: IKID; Buckle et al, 1997). Residues included in the multiple mutants Ml and M2 (A212, A223, M233. 1305, E308 and N326) are indicated. Drawn with MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt & Murphy, 1994).

Figure 2. Urea denaturation at 25 °C of GroEL(193-345) wild-type and two multiple mutants Ml and M2. The fraction of protein remaining folded for wild-type (Δ), Ml (0) and M2 (O) is represented as a function of urea concentration. The protein concentration is -10 μM. The best fitting of the experimental data to a two-state transition model using the program Kaleidagraph™ is indicated by solid lines.

Figure 3. In vitro refolding of human CyPA in the presence of various chaperones. They are: no chaperone (Spont.), 0.94 μM GroEL, 0.94 μM GroEL(193-345), 0.94 μM Ml, and 0.94 μM M2. The final concentration of CyPA is 0.94 μM. One hundred percent activity was obtained with native CyPA. Standard error bars are shown. EXAMPLES

Materials and Methods

Site-directed mutagenesis and production of proteins

Standard molecular biology methods were employed (Sambrook et al, 1989). The plasmids pRSETA GroEL(193-335) and pRSETA GroEL(193-345) are previously described (Chatellier et al. 1998). Site-directed mutagenesis was made by polymerase chain reaction (PCR) with two complementary primers following the instruction of Quickchange™ (Stratagene. USA). Mutations were confirmed by dideoxynucleotide sequencing using PRISM dye terminators (ABI. USA). Multiple mutants were obtained by multiple cycles of PCR. The overexpression and purification of minichaperones and their mutants were carried out essentially as described previously (Zahn et al . 1996). Production of GroEL was as described (Corrales & Fersht, 1996).

Denaturation experiments

The chemical denaturation was performed using urea as denaturant in 50 mM TrisΗCl, 150 mM NaCI, pH 8.2 at 25 °C. monitored at 218nm using a Jasco J-720 spectropolarimeter with cuvettes of pathlength of 0.1 cm, essentially as described previously (Golbik et al. 1998). The plots of CD signals at 218 nm versus urea concentrations were fitted to a two- state equation (Clarke & Fersht. 1993) using the program KaleidaGraph™:

(α_N + b_N[urea]) + (a_D + b_D[urea]) exp[m_D._N([urea] - [urea]₅₀o_/o)/RT] F =

1 + exp[m_D._N([urea] - [urea]₅₀o_/o)/R7]

Here F is the recorded CD signal, α_N and a_D are the intercepts and b_N and b_O are the slopes of the pre-transition and post-transition regions where proteins are in native (N) and denatured (D) states, [urea] _50% is the concentration of urea at which 50%o of the proteins is denatured; mo-_N is the slope of the transition region which reflects the sensitivity to chemical denaturant (urea); R is the gas constant and -T is the absolute temperature. DSC experiments were performed essentially as described previously (Zal n et al . 1996) The protein concentration was -100 μM in 25 mM sodium phosphate buffer, 100 mM sodium chloride. pH 7 8 The data were fitted to a non-two-state model and values for T_m, the midpoint of thermal denaturation, were obtained

Refolding Experiments

Human CyPA was denatured by incubating the protein (94 μM) in 100 mM potassium phosphate, 10 mM DTT, pH 7 0 and 8 M urea at 30 °C for at least 16 h Refolding of CyPA was initiated by diluting the urea-denatured protein 100-fold to a final concentration of 0 94 μM in 100 mM potassium phosphate. 10 mM DTT, pH 7 0 supplemented with 0 94 μM of \ aπous chaperoi > The concentration of GroEL is in terms of monomers Refolding was allowed to proceed for 2 h at 25°C Regain of CyPA activity was assayed spectrophotometπcally using a modification of a method first described by Fischer and coworkers (Fischer et al , 1984) Briefly, immediately before the assay, 50 μL of the refolding samples were diluted into 987 5 μL of assay mixture (100 mM potassium phosphate. 10 mM DTT, pH 7 0 containing 0 3 mg mL ' α-chymotrypsin) at 10^°C 12 5 μL of peptide substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanihde (dissolved m tπfluoroethanol/0 45 M LiCl at 6 mM) was then added and absorption at 390 nm was lecorded as a function of time One hundred percent activity was obtained for native CyPA, hile the spontaneous refolding in the absence of chaperone is close to zero

Results and Discussion

Design of minichaperone mutants

Sequence compaπson of E coli GroΕL(193-345) with the apical domains of 129 CpnόO proteins reveals that 31 of the E coli GroEL(193-345) residues occur with a frequency of less than 35% These residues were selected to be individually replaced by the dominant residues at the respective positions and are listed in Table 1 The selected residues are scattered over the entire domain and have side-chain solvent accessibility ranging from completely buried (M233) to more than 90% exposure (R231) No preference of their location in structure is apparent. Residues involved in substrate binding (Fenton et al, 1994; Buckle et al . 1997) are highly conserved and remain unchanged.

Stability of minichaperones and their mutants Two minichaperones were used in this study, GroEL(193-345) and GroEL(193-335), the latter is the minimal minichaperone identified to be active (Chatellier et al. 1998). The intrinsic fluorescence of both GroEL(193-335) and GroEL(193-345) from the two tyrosine residues, Tyrl99 and Tyr203. is too weak to allow accurate monitoring of denaturation induced by urea or temperature. Circular dichroism (CD) spectroscopy was, therefore, used instead, which reflects changes in helicity upon denaturation. The values for [urea]_50% are 2.8 M and 3.1 M for GroEL(193-335) (Table 2) and GroEL( 193-345) (Table 3) respectively.

For the first six mutations. GroEL(193-335) was used as parent protein (Table 2). However, its high tendency to aggregate presented severe difficulties in purifying some mutant proteins. The larger construct, GroEL( 193-345), which does not aggregate to such a degree, was thus used as parent protein for all the subsequent mutations. Except for three mutations D316M. Q319T and R322K, for which no proteins can be obtained, the thermodynamic parameters of each single mutation were deti -TNned by urea-induced equilibrium denaturation (Table 2, 3 ). The mo- values of both constructs and their respective mutants are fairly stable, with an average of 2.23±0.05 for GroEL(193-335) and 2.15±0.03 for GroEL(193-345). The standard errors in ΔΔG^ ° are 0.3 to 0.5 kcal mob¹ for GroEL(193-335) and 0.2 kcal mob¹ for GroEL(193-345), whereas the errors in ΔΔG<-~ι are much smaller, 0.05 to 0.08 kcal mob¹ for GroEL( 193-335) and 0.01 to 0.03 kcal mob¹ for GroEL(l 93-345). The inherent inaccuracy of ΔΔG» » has been reported to originate from the long linear extrapolation to the absence of denaturant (Jackson et al , 1993). Therefore, ΔΔG_| -u- was used as an accurate measure of the changes in stability upon mutation. The effect on stability of each single mutation ranges from +1.55 to -1.78 kcal mob¹, relative to the respective wild-type minichaperones (Table 2, 3).

(i) Stabilizing mutations Thirteen mutations stabilize the protein by more than 0.10 kcal mob¹. Six occur in loops (K207N, A212E, V271L. I305L, E308K and E308S). four are in β-strands (S217D. A223T, A223V and N326T), while three in α-helices (M233L, M267L and T294R). Mutations A212E. A223T, A223V, M233L and N326T each stabilize the protein by more than 1 kcal mob¹. The T_m values of these five mutants were analyzed by differential scanning calorimetry (DSC), the increases range from 1.55 to 6.78 °C (Table 4). The increases in chemical stability (ΔΔGι» ...) and thermostability (ΔT_m) correlate very well with each other (correlation coefficient of 0.97).

A212 is located in a loop between β-strands S6 and S7 and is almost completely buried. The side chain of A212 is about 4 A away from the side chains of K207 and V213, and is surrounded by potential hydrogen-bonding partners from residues T210, G21 1 , V213, 1325 and water molecules. The mutation A212E stabilizes the protein by 1.38 kcal mob¹ and 5.63 ^°C. This could be the result of the enhanced hydrophobic packing due to the introduction of one methylene group, and hydrogen bonding with the introduced carboxyl group.

A223 is the C-terminal residue of β-strand S8 and is located in the hydrophobic core. It occurs with a frequency of 7% in the Cpn60 family. A223 is surrounded by the methyl(ene) groups of 1227. A251 and 1301 , and hydrogen-bonding groups of L222, D224. K225, A250 and 1301. all within a radius of 4.5 A. The mutation A223T stabilizes the protein by 1.55 kcal mob' and 5.60 °C. As for the mutation A212E. the source of the stabilization is likely to be the increased hydrophobic packing and hydrogen-bonding interactions. Although residue A223 is nearly completely buried in the hydrophobic core, the more hydrophobic substitution A223V is 0.52 kcal mob¹ and 4.05 °C less stable than its isosteric substitution A223T.

The most stabilizing mutation is M233L, a similar-size replacement on the buried face of α- helix H8 that participates in the hydrophobic core. In the crystal structure of GroEL(191- 376) solved at 1.7 A (Buckle et al, 1997), the side chain of M233 displays two conformations, indicating intrinsic local disorder in the wild-type protein that may stabilize the protein entropically. Surrounding residues within 4.5 A, namely L221, 1227, 1230, V236, L237, L247, 1249 and L262, form a small hydrophobic cluster. Among them, 1230 and L237 have been postulated to interact with substrates (Fenton et al, 1994; Buckle et al , 1997). Though the theoretical gain in Gibbs free energy for Met -» Leu at fully buried positions is only 0.6 kcal mob¹ (Fauchere & Pliska, 1983), the mutation M233L stabilizes the protein by 1.78 kcal mob¹ and 5.90 °C.

N326 is the C-terminal residue of β-strand Sl l, with 53% of its side chain accessible to solvent. Its close neighbours within 4.5 A are mainly oxygen and nitrogen atoms from surrounding residues 1325. K327, D328 and T329. The mutation N326T stabilizes the protein by 1.35 kcal mob¹ and 6.78 °C. where hydrogen-bonding interactions may contribute to the enhanced stability.

T294 is located in α-helix H10 close to its C-terminus. and has a solvent accessibility of 17%). The mutation T294R stabilizes the protein by 0.37 kcal mob¹. Stabilization may be the result of the favorable electrostatic interaction between the helical dipole and the introduced R294. 1305 and E308 are located in the solvent-exposed segment of a mobile loop between H10 and SI 1. The region between residues 301 and 308 is the least resolved in the crystal structures of intact GroEL (Braig et al. 1995), which suggests an inherent flexibility of this region. The mutations I305L, E308K and E308S stabilize the protein by 0.69, 0.34 and 0.47 kcal mob¹ respectively. I305L and E308K are included in two multiple mutants described below. Other mutations K207N. S217D, M267L and V271L stabilize the protein by 0.27, 0.15, 0.13 and 0.15 kcal mob¹ respectively.

Generally, substituting a small hydrophobic residue in the core of a protein by another of larger size ("small-to-large") will result in destabilization (Fersht et al , 1987). Protein cores are densely packed, in order to accommodate a substituted larger side chain a reorganization of the local or even global structure is always required which, in most cases, destabilizes the protein. However, in our study, three small-to-large mutations A223T, A223V and V236L (0.58 kcal mob¹), all occurring in the protein core, are stabilizing mutations. Another stabilizing mutation occurring nearby is the similar-size substitution M233L. Strikingly, A223T, A223V, M233L and V236L are clustered in the hydrophobic core that buttresses the substrate-binding site of GroEL. (ii) Destabilizing mutations. Sixteen of the 34 single mutations destabilize the protein by more than 0.10 kcal mob¹. Seven are in loops (N206T, P208S, T210K, I270G, I270T, T299Q and M307L). three in β-strands (V213A, T329N and I333V) and six in α-helices (R231Q. E232D, A239Q, K242Q, A243S and T266K). The largest loss of stability is caused by the truncation of a buried hydrophobic residue in the core of the protein to a smaller one (V213A). The destabilizing effect is 1.55 kcal mob¹. Similarly, another truncated mutation I333V at a largely buried position destabilizes the protein by 0.95 kcal mob¹. These "large-to-small" mutations in the interior of a protein could destabilize the protein by the reduction in hydrophobicity and the introduction of one or more cavities of varying sizes (Lee, 1993; Buckle et al , 1996; Matthews. 1996).

(Hi) Neutral nvw t ions There are 5 neutral mutations with within the experimental error (0.10 kcal mob¹). These are G211 M. G21 1Q (loop between β-strands S6 and S7), F219Y (β-strand S8), T294I (α-helix H10) and Q319K (β-strand Sl l).

Ultimately, the correlation of energetic and structural responses to mutation awaits the elucidation of the three-dimensional structures of the mutant minichaperones.

Semi-rational design of highly stable multiple mutants of GroEL(193-345) Two multiple variants (Ml : A212E/A223T/M233L/I305L/E308K/N326T; M2:

A212E/A223V/M233L/I305L/E308K/N326T) were constructed, each encompassing six stabilizing mutations (Figure 1). The only difference between Ml and M2 is the residue at position 223; T223 in Ml , whereas V223 in M2. The values of [urea]_50% are 6.37 and 5.98

M for Ml and M2 respectively (Figure 2), which correspond to 6.99 and 6.15 kcal mob¹ more stable than wild-type protein, in terms of ΔΔGι-~ι» . The sum of stabilizing effects of the constituent mutations is roughly equal to the increased stability of Ml and M2 (Table

5). Despite the close proximity of some residue-pairs in the three-dimensional structure, such as A212 and N326, A223 and M233, 1305 and M308, the stabilizing effects are additive rather than co-operative. The T_m values measured by DSC are 85.66 °C for Ml and 81.26 °C for M2, 18.58 °C and 14.18 °C higher than that of wild type respectively

(Table 5). Activity of minichaperone multiple mutants

CyPA is a peptidyl-prolyl cis-trans isomerase with a molecular mass of 18 kDa. The refolding of denatured CyPA requires the assistance of GroEL (Zahn et al, 1994) or minichaperones (Zahn et al, 1996) in order to achieve a high recovery of activity. The highly stable minichaperone variants Ml and M2 demonstrate high activity in assisting the refolding of CyPA. at the same level as full-length GroEL and wild-type GroEL( 193-345) (Figure 3). Spontaneous refolding of CyPA has been reported to be about 30%> in the absence of chaperones. However, when human CyPA was subjected to denaturing condition (100 mM potassium phosphate. 10 mM dithiothreitol (DTT), pH 7.0 and 8 M urea) for at least 16 h at 30 °C. no significant spontaneous refolding upon removal of urea was observed. Therefore, the modified method described here renders the measured chaperone activity much more reliable than previously reported.

Validity of the approach Out of the 34 single mutants made in this study, 13 are stabilizing, 16 are destabilizing, while 5 neutral. It seems somewhat disappointing that the chance for obtaining stabilizing mutants is only about 38%>. However, lowering the threshold of sequence identity from the previous 35% to 20% when selecting target residues, results in an increased success rate. Among a set of 18 mutations included, there are 13 stabilizing mvt n ions. 2 neutral and only 3 destabilizing, as the majority of destabilizing mutations are of sequence identity between 20% to 35%. Thus, the success rate is increased to 72%, which undoubtedly demonstrates the validity of this approach. Nevertheless, qualitative correlation between the occurrence of a certain residue and its effect on stability (Steipe et al . 1994) has not been observed.

There are so far five CpnόO proteins isolated from habitats of extremely high temperature that are closely related to GroEL in primary sequence. The overall homology pattern of sequence alignment among the apical domain of GroEL and these thermophilic CpnόO proteins is quite similar to that among 130 CpnόO proteins. From the whole mutant pool, a subset of 20 mutations was selected whereas 3 out of 5 sequences suggest the mutations. Among them, 8 mutations are stabilizing, one is neutral, while 1 1 are destabilizing. Not surprisingly, the majority of the stabilizing mutations, especially all the five most stabilizing ones (A212E, A223T, A223V, M233L and N326T), are indicated by this "small scale" sequence alignment. Therefore, in good agreement with previous studies (Shih & Kirsch, 1995), sequence alignment of thermolabile proteins with thermostable proteins is an efficient way of designing substitutions to stabilize proteins.

58-

Table 1. Properties of residues³ selected for mutation

Residue¹³ Residue Side-chain solvent

Position Location Frequency⁰ (%) accessibility ^d (%)

N206 Loop between S6 and S7 25 26

K207 Loop between S6 and S7 12 51

P208 Loop between S6 and S7 35 82

T210 Loop between S6 and S7 22 65

G211 Loop between S6 and S7 7

A212 Loop between S6 and S7 5 9

V213 S7 J -5 1

S217 S7 8 74

F219 S8 26 9

A223 S8 7 1

R231 H8 27 91

E232 H8 28 16

M233 H8 20 0

A239 H8 22 21

K242 H8 35 77

A243 H8 26 13

T266 H9 10 14

M267 H9 18 47

1270 Loop between H9 and S 10 31 48

V271 Loop between H9 and S 10 19 4

T294 H10 8 17

T299 Loop between H10 and Sl l 30 52

1305 Loop between HI 0 and S 11 15 65

M307 Loop between H10 and Sl l 24 25

E308 Loop between H10 and SI 1 12 64

D316 Loop between H10 and SI 1 19 29

Q319 Sl l 21 46

R322 Sl l --o 45

N326 Sl l 12 53

T329 S12 16 27

1333 S12 35 18

^a Using the crystal structure of minichaperone GroEL(191-376) (PDB ID: IKID; Buckle et al., 1997). ^b The numbers refer to residue positions (Hemmingsen et al., 1988), while the letters represent amino acid residues using one-letter codes. ^c The relative frequency of occurrence of GroEL(l 93-345) residues as calculated from the sequence alignment of 130 CpnόO proteins. ^d Percentage of residue accessibility relative to that in an extended Gly-X-Gly tri-peptide

(Miller et al., 1987), calculated with a 1.4 A probe and the program AREAIMOL (CCP4,

1994). Table 2. Thermodynamic parameters" of urea equilibrium denaturation for GroEL( 193-335) and i

'"l)-N | iiroa l5(» ; „ ΛG "^<° ΛΛ ; !^

Mutation¹' (kcal mol-' M-i) (M) (kcal mol-') (kcal mol'¹)

GroEL(l 93-335) 2.18±0.10 2.81±0.02 6.1±0.2

K207N 2.37±0.27 2.93±0.03 6.9±0.5 -0.8-fc0.5

P208S 2.03±0.20 2.47±0.05 5.0±0.3 1. U0.4

T210K 2.34±0.14 2.69±0.02 6.3±0.2 -0.2±0.3

G211M 2.36±0.14 2.81±0.02 6.6±0.2 -0.5±0.3

G211Q 2.16±0.18 2.80±0.03 6.1±0.3 0.0±0.4

A212E 2.16±0.13 3.43±0.02 7.4±0.2 -1.3±0.3

^a Experiments were performed as described in Materials and Methods. mo-_N, slope of the transition r curve which reflects the sensitivity to chemical denaturant; [urea]₅o_%, concentration of urea at whic denatured; ΔG" _{N >} free energy of denaturation (ΔG_D._N) in the absence of denaturant, calculated using ΔΔG"i°_N, difference in ΔG"l°_N between wild-type GroEL(l 93-335) and any mutant; between wild-type GroEL( 193-335) and any mutant, calculated using ΔΔG{j^U™l5Mς = ^</W_D-N [^urea] <m_D._N> is the average value 2.23±0.05 for GroEL( 193-335) and its mutants. Positive values of ΔΔG"i° decreased stability whereas negative values indicate increased stability. ^b The numbers refer to residue positions, while the letters represent amino acid residues. For example, position 207 is mutated to N.

Table 3. Thermodynamic parameters of urea equilibrium denaturation for GroEL(193-345)^a and its mutants

' I

'"D-N [urea 1₅0_% Δ A ^ „ILJO_N ΔΔG » ΔΔG| i^W

Mutation (kcal mol-ι M-i) (M) (kcal mol-') (kcal mol-') (kcal mol- ')

GroEL(l 93-345) 2.12±0.09 3.12:1:0.01 6.6-1:0.2

N206T 2.28-1-0.05 2.79-L0.01 6.4-1-0.1 0.2±0.2 0.71 ±0.02

V213A 2.26-1:0.06 2.40-fcO.Ol 5.4*0.1 1.2±0.2 1 .55±0.03

S217D 2.14±0.04 3.20±0.01 6.8-1:0.1 -0.2±0.2 -0.15±0.02

F219Y 2.10±0.05 3.15±0.01 6.6±0.1 0.0±0.2 -0.06±0.01

A223T 1.96±0.05 3.84±0.01 7.5±0.1 -0.9±0.2 -1.55±0.03

A223V 2.05±0.08 3.60±0.01 l.A±0.2 -0.8±0.2 - 1.03±0.03 o

R231Q 2.28±0.08 2.95±0.01 6.7±0.1 -0.U0.2 0.37±0.02

E232D 2.17±0.04 2.79±0.01 6.1±0.1 0.5±0.2 0.71±0.02

M233L 1.80±0.04 3.95±0.01 7.1±0.1 -0.5±0.2 -1.78±0.03

A239Q 2.45±0.08 3.00±0.01 7.3±0.1 -0.7±0.2 0.26±0.02

K242Q 2.43±0.07 2.9U0.01 7. U0.1 -0.5±0.2 0.45±0.02

A243S 2.14±0.05 3.02±0.01 6.5±0.1 0.1±0.2 0.21±0.02

T266K 2.30±0.05 2.97±0.01 6.8±0.1 -0.2±0.2 0.32±0.02

M267L 2.49±0.08 3.18±0.01 7.9±0.1 -1.3±0.2 0.13±0.02

I270G 2.20±0.04 3.0U0.01 6.6-t0.1 0.0±0.2 0.24±0.02

I270T 2.15±0.03 2.90±0.01 6.2±0.1 0.4±0.2 0.47±0.02

V271L 2.0U0.04 3.27±0.01 6.6±0.1 0.0-t0.2 0.15±0.02

T294R 2.40±0.09 3.29±0.01 7.9±0.2 -1.3±0.2 0.37±0.02

Table 4. Midpoints (T_m) for thermal denaturation of GroEL(193-345) and representative stabilizing mutants determined by DSC^a

Protein T_m (-Q Δ-T_m(°C)

GroEL(193- -345) 67.08±0.02

A212E 72.71±0.07 5.63

A223T 72.68±0.03 5.60

A223V 68.63±0.03 1.55

M233L 72.98±0.03 5.90

N326T 73.86±0.01 6.78

^a Experiments were performed as described in Materials and Methods. T_m is the midpoint of thermal denaturation. Δ-T_m is the change in T_m relative to wild-type GroEL(l 93-345). calculated as &T_m = -H™" - T2

Table 5. Thermodynamic parameters" for two highly stable GroEL(l 93-345) mutants

'"D-N |urea]_5()% AG "'° ΛΛ(/_π! _N

Protein^b (kcal mol-" M-«) (M) (kcal mol-') (kcal mol-') (kcal mol-¹) (kcal mol-') CC) CC)

Ml 1.84±0.04 6.37±0.01 1 1.7±0.1 -5.U0.2 -6.99±0.02 -7.09 85.66±0.02 18.58

M2 1.78±0.04 5.98±0.01 10.6J 0.1 -4.0±0.2 -6.15±0.02 -6.57 81.26.t0.03 14.18

^a All as defined in Table 2 and Table 4. & b GroEL(l 93-345) Ml includes six mutations: A212E, A223T, M233L, I305L, E308K and N326T, while GroEL( 193-345)

M2 includes six mutations: A212E, A223V, M233L, I305L, E308K and N326T.

^C∑ΔΔG^--"^U is ^{the sum} °f ^the effects on protein stability (ΔΔGJ""¹** ) of all constituent single mutations.

References

Altamirano MM. Garcia C, Possani LD, Fersht AR. 1999. Oxidative refolding chromatography: folding of the scorpion toxin Cn5 [In Process Citation]. Nat Biotechnol 77:187-191.

Altamirano MM, Golbik R, Zahn R. Buckle AM, Fersht AR. 1997. Refolding chromatography with immobilized mini-chaperones. Proc Natl Acad Sci U S A 94:3516- 3578.

Ben-Zvi AP, Chatellier J. Fersht AR, Goloubinoff P. 1998. Minimal and optimal mechanisms for GroE-mediated protein folding. Proc Natl Acad Sci U S A 95: 15275-

15280.

Braig K. Adams PD. Brunger AT. 1995. Conformational variability in the refined structure of the chaperonin GroEL at 2.8 A resolution [see comments]. Nat Struct Biol 2: 1083-1094. Buckle AM, Cramer P, Fersht AR. 1996. Structural and energetic responses to cavity- creating mutations in hydrophobic cores: observation of a buried water molecule and the hydrophilic nature of such hydrophobic cavities. Biochemistry 55:4298-4305. Buckle AM. Zahn R, Fersht AR. 1997. A structural model for GroEL-polypeptide recognition. Proc Natl Acad Sci USA 94:251 -3515.

CCP4. 1994. The CCP4 Suite: Programs for Protein Crystallography. Ada Cryst. -050:760-

763.

Chatellier J. Hill F, Lund PA. Fersht AR. 1998. In vivo activities of GroEL minichaperones. Proc Natl Acad Sci USA 95:9861-9866. Clarke J, Fersht AR. 1993. Engineered disulfide bonds as probes of the folding pathway of barnase: increasing the stability of proteins against the rate of denaturation. Biochemistry 32:4322-4329.

Corrales FJ, Fersht AR. 1996. Kinetic significance of GroEL14 (GroES )₂ complexes in molecular chaperone activity. Fold Des 1:265-213. Fauchere J-L, Pliska V. 1983. Hydrophobic parameters II of amino acid side-chains from the partitioning of N-acetyl-amino-acid amides. Eur. J. Med. Chem. 18:369-315. Fenton WA, Kashi Y, Furtak K, Horwich AL. 1994. Residues in chaperonin GroEL required for polypeptide binding and release [see comments]. Nature -377:614-619. Fersht AR, Leatherbarrow RJ, Wells TN. 1987. Structure-activity relationships in engineered proteins: analysis of use of binding energy by linear free energy relationships.

Biochemistry 2(5:6030-6038.

Fischer G, Bang H, Berger E, Schellenberger A. 1984. Conformational specificity of chymotrypsin toward proline-containing substrates. Biochim Biophys Ada 791:11-91.

Gething MJ. Sambrook J. 1992. Protein folding in the cell. Nature 355:33-45.

Golbik R, Zahn R, Harding SE, Fersht AR. 1998. Thermodynamic stability and folding of

GroEL minichaperones. J Mol Biol 27-5:505-515.

Hemmingsen SM, Woolford C, van der Vies SM, Tilly K, Dennis DT, Georgopoulos CP, Hendrix RW. Ellis RJ. 1988. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333:330-334.

Jackson SE. Moracci M, elMasry N, Johnson CM. Fersht AR. 1993. Effect of cavity- creating mutations in the hydrophobic core of chymotrypsin inhibitor 2. Biochemistry

52: 1 1259- 1 1269. K-raulis PJ. 1991. Molscript: a program to produce both detailed and schematic plots of protein structure. J- Appl. Crystallogr 24:946-950.

Lee B. 1993. Estimation of the maximum change in stability of globular proteins upon mutation of a hydrophobic residue to another of smaller size. Protein Sci 2:733-738.

Matthews BW. 1996. Structural and genetic analysis of the folding and function of T4 lysozyme. R- NE-S J 70:35-41.

Merritt EA. Murphy MEP. 1994. Raster3D version 2.0. A program for photorealistic molecular graphics. Ada Crystallogr D50:869-873.

Miller S, Janin J, Lesk AM, Chothia C. 1987. Interior and surface of monomeric proteins. J

Mol Biol 196:641-656. Nikolova PV, Henckel J. Lane DP. Fersht AR. 1998. Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability. Proc Natl Acad Sci U S A

95: 14675-14680.

Regan L, Serrano L, Sali A, Horovitz A, Wilson C. 1996. Paper Alert. Fold Des 7:R1 15-

1 19. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: laboratory manual. Cold

Spring Harbor, New York: Cold Spring Harbor Laboratory. Serrano L, Day AG, Fersht AR. 1993. Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability. J Mol Biol 253:305-312.

Shih P, Kirsch JF. 1995. Design and structural analysis of an engineered thermostable chicken lysozyme. Protein Sci 4:2063-2072.

Steipe B, Schiller B, Pluckthun A, Steinbacher S. 1994. Sequence statistics reliably predict stabilising mutations in a protein domain. J. Mol. Biol 2-70:188-192.

Zahn R, Buckle AM, Perrett S, Johnson CM, Corrales FJ, Golbik R, Fersht AR. 1996. Chaperone activity and structure of monomeric polypeptide binding domains of GroEL [see comments]. Proc Natl Acad Sci USA 93:15024-15029.

Zahn R. Spitzfaden C. Ottiger M, Wuthrich K, Pluckthun A. 1994. Destabilization of the complete protein secondary structure on binding to the chaperone GroEL. Nature 5-55:261- 265.

SEOUENCE LISTING

SEQ ID NO. 1 - amino acid sequence of E. coli GroEL - Accession No. AAC77103

1 MAAKDVKFGN DARVK LRGV NVLADAVKVT LGPKGRNWL DKSFGAPTIT KDGVSVAREI

61 E EDKFENMG AQMVKEVASK ANDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI

121 DKAVTAAVEE LKALSVPCSD SKAIAQVGTI SA SDETVGK LIAEAMDKVG KEGVITVEDG

181 TGLQDELDW EGMQFDRGYL SPYFINKPET GAVELESPFI LLADKKISNI REMLPVLEAV

241 AKAGKPLL: I EDVEGEALA TLWNTMRGI VKVAAVKAPG FGDRRKAMLQ DIATLTGGTV

301 ISEEIGMELE KATLEDLGQA KRWINKDTT TIIDGVGEEA AIQGRVAQIR QQIEEATSDY

361 DREKLQERVA KLAGGVAVIK VGAATEVEMK EKKARVEDAL HATRAAVEEG WAGGGVALI

421 RVASKLADLR GQNEDQNVGI KVALRAMEAP LRQIVLNCGE EPSWANTVK GGDGNYGYNA

481 ATEEYGNMID MGILDPTKVT RSALQYAASV AGLMITTECM VTDLPKNDAA DLGAAGGMGG

541 MGGMGGMM

SEQ ID NO. 2 - nucleotide sequence of £ coli GroEL - Accession No. AE000487 (nucleotides 2515-4161)

ATGGCA

GCTAAAGACG TAAAATTCGG TAACGACGCT CGTGTGAAAA TGCTGCGCGG

CGTAAACGTA

CTGGCAGATG CAGTGAAAGT TACCCTCGGT CCAAAAGGCC GTAACGTAGT

TCTGGATAAA

TCTTTCGGTG CACCGACCAT CACCAAAGAT GGTGTTTCCG TTGCTCGTGA

AATCGAACTG GAAGACAAGT TCGAAAATAT GGGTGCGCAG ATGGTGAAAG AAGTTGCCTC

TAAAGCAAAC

GACGCTGCAG GCGACGGTAC CACCACTGCA ACCGTACTGG CTCAGGCTAT

CATCACTGAA

GGTCTGAAAG CTGTTGCTGC GGGCATGAAC CCGATGGACC TGAAACGTGG

TATCGACAAA

GCGGTTACCG CTGCAGTTGA AGAACTGAAA GCGCTGTCCG TACCATGCTC

TGACTCTAAA

GCGATTGCTC AGGTTGGTAC CATCTCCGCT AACTCCGACG AAACCGTAGG

TAAACTGATC

GCTGAAGCGA TGGACAAAGT CGGTAAAGAA GGCGTTATCA CCGTTGAAGA

CGGTACCGGT

CTGCAGGACG AACTGGACGT GGTTGAAGGT ATGCAGTTCG ACCGTGGCTA

CCTGTCTCCT

TACTTCATCA ACAAGCCGGA AACTGGCGCA GTAGAACTGG AAAGCCCGTT

CATCCTGCTG

GCTGACAAGA AAATCTCCAA CATCCGCGAA ATGCTGCCGG TTCTGGAAGC

TGTTGCCAAA

GCAGGCAAAC CGCTGCTGAT CATCGCTGAA GATGTAGAAG GCGAAGCGCT

GGCAACTCTG

GTTGTTAACA CCATGCGTGG CATCGTGAAA GTCGCTGCGG "TΛAAGCACC

GGGCTTCGGC

GATCGTCGTA AAGCTATGCT GCAGGATATC GCAACCCTGA CTGGCGGTAC

CGTGATCTCT

GAAGAGATCG GTATGGAGCT GGAAAAAGCA ACCCTGGAAG ACCTGGGTCA

GGCTAAACGT

GTTGTGATCA ACAAAGACAC CACCACTATC ATCGATGGCG TGGGTGAAGA

AGCTGCAATC

CAGGGCCGTG TTGCTCAGAT CCGTCAGCAG ATTGAAGAAG CAACTTCTGA

CTACGACCGT

GAAAAACTGC AGGAACGCGT AGCGAAACTG GCAGGCGGCG TTGCAGTTAT

CAAAGTGGGT

GCTGCTACCG AAGTTGAAAT GAAAGAGAAA AAAGCACGCG TTGAAGATGC

CCTGCACGCG

ACCCGTGCTG CGGTAGAAGA AGGCGTGGTT GCTGGTGGTG GTGTTGCGCT

GATCCGCGTA GCGTCTAAAC TGGCTGACCT GCGTGGTCAG AACGAAGACC AGAACGTGGG

TATCAAAGTT

GCACTGCGTG CAATGGAAGC TCCGCTGCGT CAGATCGTAT TGAACTGCGG

CGAAGAACCG

TCTGTTGTTG CTAACACCGT TAAAGGCGGC GACGGCAACT ACGGTTACAA

CGCAGCAACC

GAAGAATACG GCAACATGAT CGACATGGGT ATCCTGGATC CAACCAAAGT

AACTCGTTCT

GCTCTGCAGT ACGCAGCTTC TGTGGCTGGC CTGATGATCA CCACCGAATG

CATGGTTACC

GACCTGCCGA AAAACGATGC AGCTGACTTA GGCGCTGCTG GCGGTATGGG

CGGCATGGGT

GGCATGGGCG GCATGATGTA A

Claims

1. A GroEL chaperone polypeptide. or homologue thereof, or fragment thereof having protein refolding activity, comprising one or more amino acid modifications at any one of amino acid residues 207, 212. 217, 223, 233, 267, 271, 294, 305, 308 and 326 of the GroEL amino acid sequence shown as SEQ I.D. No. 1 or their equivalent positions in other homologous chaperone polypeptides.

2. A polypeptide according to claim 1 wherein said modifications are selected from Lys207→Asn, Ala212→Glu. Ser217→Asp, Ala223→Thr, Ala223→Val, Met233-»Leu, Met267- Leu. Val271→Leu. Val271→Ser, Thr294→Arg, Ile305→Leu and Glu308→Lys, Asn326— - Thr, and their equivalents in other homologous chaperone polypeptides.

3. A polypeptide according to claim 2 wherein said modifications are selected from [Ala212→Glu, Ala223→Thr, Met233→Leu, Ile305→Leu. Glu308→Lys and Asn326→Thr], and [Ala212→Glu, Ala223→Val, Met233→Leu, Ile305→Leu, Glu308— ->Lys and Asn326-»Thr], and their equivalents in other homologous chaperone polypeptides.

4. A polypeptide according to any one of claims 1 to 3 consisting essentially of amino acids 193 to 335, 191 to 337. 191 to 345 or 191 to 376 of GroEL or the equivalent residues of homologous chaperone polypeptides.

5. A polypeptide according to any one of the preceding claims which, when in solution, remains monomeric and has the ability to refold, reactivate or recondition proteins.

6. A polypeptide according to any one of the preceding claims fused to a heterologous polypeptide.

7. A polypeptide according to any one of the preceding claims immobilised to a solid phase.

8. A recombinant polypeptide according to any one of the preceding claims.

9. A nucleic acid molecule encoding a polypeptide according to any one of the preceding claims.

10. A vector comprising a nucleic acid according to claim 9, optionally operably linked to a regulatory sequence capable of directing expression of said nucleic acid in a suitable host cell.

11. A host cell comprising a nucleic acid according to claim 9 or a vector according to claim 10.

12. A method of making a polypeptide as defined in any one of claims 1 to 8 comprising transforming a host cell with a nucleic acid encoding said polypeptide, culturing the transformed cell and expressing said polypeptide.

13. A pharmaceutical formulation comprising a polypeptide according to any one of claims 1 to 8 or a nucleic acid according to claim 9 together with a pharmaceutically acceptable diluent, carrier or excipient.

14. A polypeptide according to any one of claims 1 to 8 or a nucleic acid according to claim 9 for use in therapy.

15. Use of a polypeptide according to any one claims 1 to 8 in the manufacture of a medicament for the treatment of disease associated with protein/polypeptide structure.

16. A method of reconditioning a molecule comprising contacting said molecule with the chaperone polypeptide of any one of claims 1 to 8.

17. A method according to claim 16, wherein the molecule is subjected to inactivation or denaturation prior to contacting with said chaperone polypeptide.

18. A method according to claim 16 or 17 wherein the chaperone polypeptide is immobilised to a solid phase.

19. A method according to claim 18 wherein solid phase is a chromatographic matrix and the contacting of the molecule and chaperone polypeptide is carried out by applying the molecule to the top of a bed of the matrix packed in a column and then eluting the molecule through the column.

20. Use of a polypeptide according to any one of claims 1 to 8 for altering the structure of a molecule.

21. Use according to claim 20, wherein the molecule is a protein or polypeptide and the alteration in structure is by folding, unfolding or refolding.

22. Use according to claim 20 or 21 wherein the stoichiometry between the chaperone polypeptide and the molecule being altered is about 1 :1.

23. Use of a polypeptide according to any one of claims 1 to 8 in purifying or increasing the yield, specific activity and/or quality of biological rr le-.ules.

24. A kit for reconditioning or refolding a molecule comprising a polypeptide according to any one of claims 1 to 8 immobilised to a solid phase and a container for holding said solid phase polypeptide.

25. Use of a chaperone polypeptide as defined in any one of claims 1 to 8 in the production of a protein or polypeptide by recombinant means, wherein the said chaperone polypeptide is co-expressed with the protein or polypeptide thereby to improve the yield, specific activity and/or quality of the protein or polypeptide.

26. An antibody reactive against a polypeptide as defined in any one of claims 1 to 8.

27. An antibody as claimed in claim 26 for use in therapy.

28. Use of an antibody as claimed in claim 27 in the manufacture of a medicament for the treatment of disease associated with protein/polypeptide structure.

29. A method of treating a human or animal patient suffering from a disease associated with protein/polypeptide structure which method comprises administering to a patient an effective amount of a polypeptide according to any one of claims 1 to 8.

30. A method of treating a human or animal patient suffering from a disease associated with protein/polypeptide structure which method comprises administering to a patient an effective amount of an antibody to claim 26.