Conus protein disulfide isomerase

TECHNICAL FIELD

[0003] The invention relates generally to biotechnology and, more specifically, to protein disulfide isomerases from Conus snails, nucleic acid sequences encoding the Conus protein disulfide isomerases and to methods for using the nucleic acid or protein sequences for folding disulfide-containing proteins.

BACKGROUND OF THE INVENTION

[0004]

Conus
is a genus of predatory marine gastropods (snails) which envenomate their prey. Venomous cone snails use a highly developed injection apparatus to deliver their cocktail of toxic conotoxins into their prey. In fish-eating species such as Conus magus, the cone detects the presence of the fish using chemosensors in its siphon and, when close enough, extends its proboscis and impales the fish with a hollow harpoon-like tooth and injects the venom into the fish. This injection immobilizes the fish and enables the cone snail to wind it into its mouth via the proboscis. For general information on Conus and their venom, see Coleman, N.(2nd ed., 1992. Ure Smith Press, Sydney, Australia (ISBN 0 7254 0885 5)); or website address http://grimwade.biochem.unimelb.edu.au/cone/.

[0005] Prey capture is accomplished through a sophisticated arsenal of peptides which target specific ion channel and receptor subtypes. Each Conus specie's venom appears to contain a unique set of 50-200 peptides. The composition of the venom differs greatly between species and between individual snails within each species, each optimally evolved to paralyze its prey. The active components of the venom are small peptide toxins, typically 12-30 amino acid residues in length, and are typically highly constrained peptides due to their high density of disulfide bonds.

[0006] The venoms consist of a large number of different peptide components that, when separated, exhibit a range of biological activities. When injected into mice, they elicit a range of physiological responses from shaking to depression. The paralytic components of the venom that have been the focus of recent investigation are the α-, ω- and μ-conotoxins. All of these conotoxins are believed to act by preventing neuronal communication, but each targets a different aspect of the process to achieve this action. Each venom component has a very specific pharmacologic target. For example, a linkage has been established between α-, αA- and ψ-conotoxins and the nicotinic ligand-gated ion channel; ω-conotoxins and the voltage-gated calcium channel; μ-conotoxins and the voltage-gated sodium channel; δ-conotoxins and the voltage-gated sodium channel; κ-conotoxins and the voltage-gated potassium channel; conantokins and conodynes and the ligand-gated glutamate (NMDA) channel. (Olivera et al., 1985; Olivera et al., 1990.) The pharmacological specificity of the conotoxins makes them attractive for drug development for a variety of therapeutic applications, including neurological and cardiovascular disorders.

[0007] A characteristic structural feature of conotoxins is a large number of posttranslational modifications, in particular disulfide bridges. The primary function of disulfide bonds appears to be stabilization ofthe structure. Conotoxins are grouped into families, based upon the number and arrangement of disulfide bonds. For example, two disulfide-containing α-conotoxins contain the cysteine pattern, CC—C—C, with disulfides between the 1st and 3rd, 2nd and 4th cysteines. Three disulfide-containing ω- and δ-conotoxins share the native cysteine pattern, C—C—CC—CC, whereas μ-conotoxins share the common cysteine pattern, CC—C—C—CC. For native ω-, δ-conotoxins, the 1st and 4th, 2nd and 5th and 3rd and 6th cysteines are connected; for native μ-conotoxins, the 1st and 4th, 2nd and 5th and 3rd and 6th cysteines are connected by disulfide bonds. The correct pairing of disulfides in the native conotoxins is a prerequisite for maintaining their biological activity. The disulfide bridges are formed in a process of oxidative pairing of the cysteine residues.

[0008] Conotoxins are naturally synthesized as precursors in cells (Woodward et al., 1990; Colledge et al., 1992). For all conotoxins, the precursors share a similar organization: an N-terminal signal sequence, a propeptide region and a C—terminal cysteine-rich toxin region. Each family of conotoxins is characterized by a highly conserved signal sequence, a moderately conserved propeptide region and an almost random toxin region that contains a conserved cysteine framework.

[0009] Propeptides have been shown in many biological systems to assist in the oxidative folding of polypeptides. Examples of those studies are summarized in Table 1. Folding kinetics and yields can be significantly improved when oxidation of cysteine-rich peptides is carried out using the propeptide. In the case of proguanylin, a peptide containing two disulfide bridges, folding yields improved from 7%, using mature peptide, to 95%, using the propeptide (Schulz et al., 1999). Similar studies on the guanylyl cyclase-activating peptide, GCAP-II, showed that two amino acids in the N-terminal fragment of the propeptide were directly involved in the enhancement of peptide folding (Hidaka et al., 2000). For oxidative folding of bovine pancreatic trypsin inhibitor (BPTI), the propeptide substantially increased the folding yields and the kinetics of folding through an additional N-terminal cysteine residue present in the propeptide fragment. It thus appears that propeptides can facilitate oxidative folding of polypeptides.

1TABLE 1Summary of intramolecular and intermolecular factorsinfluencing the oxidative folding of polypeptides.FactorsExamples of polypeptidesPropeptide-assistedMacrophage inhibitory cytokine-1 MIC-1 (Fairlie et al., 2001), Nerveoxidative foldinggrowth factor hNGF (Rattenholl et al., 2001), Prouroguanylin, GCAP(Hidaka et al., 1998; Schulz et al., 1999; Hidaka et al., 2000), pancreatictrypsin inhibitor BPTI (Weissman and Kim, 1992)ChaperonesHsp70 - binding to early folding intermediates (BiP/GRP78, GRP170),Hsp70/hsp40Hsp40 - cochaperones regulating Hsp70 (Sec63p, DnaJ), Hsp90 -Calreticulin/calnexingeneral chaperones (GRP94), Hsp25 (small heat-shock proteins withsingle Cys residues), Lectins - quality control of folding (calnexin,calreticulin) immunophilins - isomerization of prolines (cyclophilin,FKPB13) (Gething, 1997; 1999)Disulfide isomerasesPDI (Freedman et al., 1994; Gilbert, 1997)and otherErp72, CaPB1, CaPB2 (Rupp et al., 1994)oxido-reductasesEro1p (Tu et al., 2000)Erv2 (Servier et al., 2001)

[0010] However, not all propeptides have been shown to increase the folding yields and/or the kinetics of folding. For example, co-conotoxin MVIIA and insulin-like growth factor (IGF) are two reported examples where a propeptide did not have a direct effect on oxidative folding. Studies by Price-Carter and Goldenberg (Price-Carter et al., 1996b) suggested that the propeptide sequence neither increased folding yields nor enhanced the kinetics of folding of ω-MVIIA. While, mature ω-MVIIA folds with relatively high yields, using the propeptide of IGF did not facilitate folding. In the case of IGF, the propeptide, likewise, did not facilitate folding.

[0011] Taken together, these studies demonstrate examples where the propeptide is very important in determining folding properties of polypeptides, as well as examples where a propeptide is not directly involved in the folding mechanism. In addition to the possible role played by propeptides, a number of other molecules are known to regulate the folding pathway of peptides in order to increase the kinetics and yields of properly folded forms.

[0012] Molecular chaperones comprise a large number of proteins that are specialized as folding assistants. Their general function is to prevent the aggregation and precipitation of nascent polypeptides and folding intermediates. These chaperones are localized in the cytoplasm and in the endoplasmic reticulum (“ER”) and bind to different folding species with relatively low specificity. The ER is the main protein-folding compartment where a majority of chaperones are involved in folding, quality control and translocation of polypeptides. Since the ER is also the only compartment where oxidative folding occurs, chaperones in the ER play a prominent role in the oxidative folding of proteins. For example, BiP, a member of the Hsp70 chaperone family, was recently shown to cooperate with protein disulfide isomerase in the oxidative folding of antibodies (Mayer et al., 2000). Some examples of molecular chaperones are summarized in Table 1.

[0013] The oxidative folding of polypeptides in vivo is catalyzed by protein disulfide isomerase (PDI), which can act as both a folding catalyst and as a molecular chaperone. The activity of this enzyme was originally discovered in rat liver, but since then it has been documented in a variety of different species. PDI belongs to a group of protein-thiol oxidoreductase enzymes, which contain thioredoxin domains. A typical PDI molecule consists of two similar thioredoxin-like domains. These domains contain the Cys-Gly-His-Cys (CGHC) (SEQ ID NO:19) redox active site. The C-terminal region of PDI has an additional domain with an ER retention signal sequence. However, there are many different classes of PDIs which are distinguished based upon their thioredoxin domain arrangement and composition as summarized in (McArthur, A. G. et al., Mol. Biol. Evol. 18(8) 1455-63, 2001).

[0014] PDI catalyzes protein thiol-disulfide exchange reactions using the thioredoxin CGHC (SEQ ID NO:19) redox active site. The enzyme contains two CGHC (SEQ ID NO:19) motifs, a low affinity peptide binding site and a KDEL (SEQ ID NO:20) endoplasmic reticulum retrieval signal. PDI is also characterized by a large number of low affinity/high capacity calcium binding sites. The oxidoreductase activity of PDI is mediated by the pair of Cys residues in the active site. These Cys residues can be easily reduced to thiols, or oxidized to a disulfide, depending on the redox potential and relative concentration of substrates and products in the ER. Moreover, PDI was also shown to be sufficient for promoting oxidative folding, even in the absence of glutathione, a molecule primarily responsible for maintaining the oxidative environment of the ER (Tu et al., 2000). In addition to its catalytic role in oxidative folding, PDI can also function as a molecular chaperone. PDI was found to facilitate folding of proteins lacking disulfides, such as rhodanase or glyceraldehyde-3-phosphate dehydrogenase. This dual function of PDI was recently characterized during the oxidative folding of proinsulin (Winter et al., 2002). In the proinsulin study, PDI increased the rate of oxidative folding and prevented proinsulin aggregation.

[0015] Since PDI has been found in bacteria, fungi, plants, invertebrate and vertebrate animals, we sought to determine if Conus snails have also utilized this enzyme to produce conotoxins. Because Conus species produce a large number of disulfide-rich proteins in their venom, a need exists in the art to identify the nucleic acid sequences encoding Conus protein disulfide isomerases, to identify the sequences of Conus protein disulfide isomerases, and to use the nucleic acids or proteins in the folding of disulfide-containing proteins.

SUMMARY OF THE INVENTION

[0016] The invention relates to protein disulfide isomerases from Conus snails, to the nucleic acid sequences encoding the Conus protein disulfide isomerases, and to a method for using the nucleic acid or protein sequences for folding of disulfide-containing proteins.

[0017] Thus, one aspect of the invention relates to the amino acid sequence of a C. textile protein disulfide isomerase or PDI with at least 95% identity with the amino acid sequence set forth in SEQ ID NO:2 which has protein disulfide isomerase activity. The amino acid sequence of C. textile protein disulfide isomerases are set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8.

[0018] In another aspect, the invention features a substantially pure preparation of a C. textile protein disulfide isomerase. In preferred embodiments, the protein disulfide isomerase includes an amino acid sequence which is at least 57%, preferably at least 65%, preferably at least 75%, preferably at least 85%, and more preferably at least 90% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8, or any combination thereof.

[0019] Another aspect of the invention relates to a nucleic acid encoding a C. textile protein disulfide isomerase or a nucleic acid encoding a protein disulfide isomerase having at least 85%, preferably at least 90%, and more preferably at least 95% identity with the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8. A preferred nucleotide sequence of the nucleic acid is set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or any combination thereof.

[0020] Another aspect of the invention relates to a recombinant or isolated nucleic acid having at least 50%, preferably at least 65%, preferably at least 85%, and more preferably at least 95% identity with the sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.

[0021] Another aspect of the invention relates to vectors containing the protein disulfide isomerase encoding nucleic acid. The isolated nucleic acid encoding a protein disulfide isomerase may be included in a vector, such as a vector that is capable of directing the expression of the protein encoded by the nucleic acid in a vector-containing cell. The isolated nucleic acid in the vector can be operatively linked to a promoter, for example, a promoter that is capable of overexpressing the protein disulfide isomerase, or that is capable of expressing the protein disulfide isomerase in a conditional manner. The vector may include one or more of the following: a selectable marker, an origin of replication, or other sequences known in the art. The isolated nucleic acid encoding a protein disulfide isomerase, or a vector including this nucleic acid, may be contained in a cell, such as a bacterial, mammalian, or yeast cell.

[0022] Another aspect of the invention relates to host cells containing a vector capable of directing expression of a protein disulfide isomerase encoding a nucleic acid. Another aspect of the invention relates to host cells containing an expression cassette with the protein disulfide isomerase encoding a nucleic acid sequence and an expression cassette with a nucle~ic acid sequence encoding a disulfide-containing protein which is to be expressed and folded. Such disulfide-containing proteins include conotoxins.

[0023] In another aspect, the invention relates to a method of increasing disulfide bond formation in a protein (for example, a conotoxin, involving expressing the protein in a host cell that also expresses an isolated nucleic acid that encodes a protein disulfide-isomerase. In another embodiment, the protein disulfide-isomerase polypeptide is derived from a Conus species. In another embodiment, the protein is a conotoxin.

[0024] Another aspect of the invention relates to the use of a protein disulfide isomerase for the folding of disulfide-rich proteins, where the PDI increases the rate or yield of properly folded disulfide-rich proteins. Such use includes in vitro oxidative folding reactions.

[0025] Another aspect of the invention relates to a substantially pure antibody, such as a monoclonal or polyclonal antibody, that specifically recognizes and binds a protein disulfide-isomerase polypeptide derived from a Conus species, for example, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]
FIG. 1 is a schematic representation of the general biosynthetic pathway of conotoxins. Abbreviations: S, cysteine in the reduced form; ER, endoplasmic reticulum; MAO, monoamine oxidase; P4H, prolyl 4-hydroxylase; X, other posttranslational modification.

[0027]
FIG. 2 is an alignment of PDIs from Conus textile (Ct-PDI) (SEQ ID NO:2), silkworm (SEQ ID NO:9), sea urchin (SEQ ID NO:10), rat (SEQ ID NO:11), human (SEQ ID NO:12), Drosophila (Fly-PDI) (SEQ ID NO:13), and C. elegans (SEQ ID NO:14). This figure indicates the nonvariant amino acids by the presence of a * under that amino acid position.

[0028]
FIG. 3 is an alignment of the Conus textile Protein Disulfide Isomerase (Ct-PDI) and three isoforms. The PDI isolated from Conus textile (SEQ ID NO:2) is aligned with the three isoforms, texl (SEQ ID NO:4), tex2 (SEQ ID NO:6) and tex3 (SEQ ID NO:8), isolated from the same species. Identical amino acids are indicated by a “*” under the amino acid position.

[0029]
FIGS. 4A and 4B are an electrophoretic analysis of proteins from a Conus textile venom duct. FIG. 4A) The venom duct was dissected from C. textile and immediately divided into four equal portions. The proximal venom bulb is a muscle and does not directly participate in a production of conotoxins. FIG. 4B) The 55 kDa PDI is one of the predominant proteins from the snail's venom duct (lanes 1-4), as was confirmed by the Edman's sequencing. Lane 5 is the reference—bovine PDI.

[0030]
FIGS. 5A-5D are the steady-state distributions of the folding species for α-GI and δ-PVIA (FIGS. 5A and 5B) compared to their prosequences, pro-GI and pro-PVIA (FIGS. 5C and 5D). Linear and correctly folded forms are denoted as L and N, respectively. The α-GI and pro-GI were folded at 22° C. in 0.1 M Tris/HCl containing 1 mM EDTA, 0.5 mM GSSG and 5 mM GSH and the steady state was observed after 15 minutes of reaction. The δ-PVIA and pro-PVIA were folded at 0° C. in 0.1 M Tris/HCl containing 1 mM EDTA, 1 mM GSSG and 2 mM GSH and the steady state was observed after 16 hours of reaction. The top chromatograph in each panel shows the initial conditions, the middle chromatograph shows intermediate folding species and the bottom chromatograph shows the final folding species.

[0031]
FIG. 6 shows the effects of the propeptide on the stability of native α-GI and δ-PVIA. Changes in accumulation of the native forms for α-GI and δ-PVIA (black bars) and their prosequences (open bars) are shown. The percentage of the native form accumulation was averaged from three separate folding experiments and the standard deviation is marked. Folding experiments were performed for 2 hours in 0.1 M Tris/HCl, pH 8.7, 1 mM EDTA at 20 μM peptide concentration (α-GI and pro-GI) and 16 hours in 0.1 M Tris/HCl, pH 7.5, 1 mM EDTA at 10 μM peptide concentration (δ-PVIA and pro-PVIA).

[0032]
FIGS. 7A-7D show the kinetics of the PDI-catalyzed folding of αGI and proGI compared to the uncatalyzed reaction. FIG. 7A shows the kinetics for folding of αGI in the absence of PDI; FIG. 7B shows the kinetics for folding of αGI in the presence of bovine PDI; FIG. 7C shows the kinetics for folding of proGI in the absence of PDI; and FIG. 7D shows the kinetics for folding of proGI in the presence of bovine PDI. The nonnative form is represented by open circles, the linear form is represented by open boxes and the native form is represented by filled circles.

[0033]
FIG. 8 shows A) folding kinetics for the PDI-catalyzed and uncatalyzed oxidation of α-GI and pro-GI. The folding reactions were carried out at 0° C. in 0.1 M Tris/HCl, pH 7.5, containing 1 mM EDTA, 0.1 mM GSSG and 2 μM PDI. The filled and open circles denote native and linear forms, respectively, and the open squares represent other folding species. The experimental points were analyzed by single exponential curve fit and the kapp values for appearance of native (only in the presence of PDI) and disappearance of linear form (in the presence or absence of PDI) were calculated. For appearance of native form in the uncatalyzed reaction, we did not determine rate constants, as the points did not fit to the exponential curve. B) shows the half times for PDI-catalyzed and uncatalyzed folding of α-GI and pro-GI. The open bars represent values for the linear form and the black bars represent values for native form.

[0034]
FIGS. 9A-9D show chromatographs of the folding species. FIG. 9A is a chromatograph of αGI in the absence of PDI. FIG. 9B is a chromatograph of αGI in the presence of bovine PDI. FIG. 9C is proGI in the absence of PDI. FIG. 9D is a chromatograph of proGI in the presence of bovine PDI. The chromatographs represent the folding species present in the folding reaction after ten and 15 minutes, FIGS. 9B and 9D and 9A and 9C, respectively. “L” represents the linear form and the native form is indicated by an “N” above the appropriate peak.

[0035]
FIG. 10 shows chromatographs of the folding species. The chromatographs show the HPLC profiles of folding species of αGI catalyzed by purified C. textile PDI. The folding reactions were performed in Tris.HCl (pH 7.5) containing 1 mM EDTA and 0.1 M GSSG for 30 minutes at 0° C. α-conotoxin GI was used as the substrate at 20 μM concentration. The linear (L) and native (N) forms are marked.

[0036]
FIG. 11 shows PCR amplification of PDI from cDNA prepared from various Conus species. Lane 1 shows the PCR amplification product from C. omaria. Lane 2 shows the PCR amplification product from C. betulinus. Lane 3 shows the PCR amplification product from C. consors. Lane 4 shows the PCR amplification product from C. aurisiacus. Lane 5 shows the PCR amplification product from C. stercusmuscarum. Lane 6 shows the PCR amplification product from C. textile. Lane 7 shows the molecular weight markers, a 1 Kb ladder.

DETAILED DESCRIPTION OF THE INVENTION

[0037] As used herein, the terms “conotoxin” and “conotoxin polypeptide” comprise conantokin peptides, conantokin peptide derivatives, conotoxin peptides and conotoxin peptide derivatives. Conotoxins are typically derived from the venom of Conus snails and may include one or more amino acid substitutions, deletions and/or additons. These peptides maybe referred to in the literature as conotoxins, conantokins or conopeptides. The conotoxin may be produced by methods, such as in vitro translation, in vitro transcription and translation, recombinant expression systems, and chemical synthesis.

[0038] As used herein, the phrase “disulfide-rich peptide” contemplates a polypeptide or protein having two or more possible disulfide bonds. Examples ofdisulfide-richpeptides include, but are not limited to, spider toxins, conotoxins, antibodies and fragments thereof, such as fragments of conotoxins and Fab fragments. Disulfide linkages can be formed between cysteine residues ofthe same or different polypeptides.

[0039] As used herein, “substantially pure” means a preparation which is at least 60% by weight (dry weight) of the compound of interest, for example, a protein disulfide isomerase or a disulfide-rich peptide. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the compound of interest. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0040] As used herein, an “isolated nucleic acid” means a nucleic acid that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant nucleic acid which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (for example, a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant nucleic acid which is part of a hybrid gene encoding additional polypeptide sequences.

[0041] As used herein, a “substantially identicar′ polypeptide sequence means an amino acid sequence which differs from a reference sequence only by conservative amino acid substitutions, for example, substitution of one amino acid for another ofthe same class (for example, valine for glycine, arginine for lysine, etc.) or by one or more nonconservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function ofthe polypeptide (assayed, for example, as described herein). Preferably, such a sequence is at least 73%, more preferably at least 85%, and most preferably at least 95% substantially identical at the amino acid level to the sequence used for comparison. The invention encompasses polypeptide sequences being 73-99% substantially identical to the amino acid sequences set forth is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8, or any combination thereof.

[0042] As used herein, a “substantially identical” nucleic acid means a nucleic acid sequence which encodes a polypeptide differing only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (for example, valine for glycine, arginine for lysine, etc.) or by one or more nonconservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the polypeptide (assayed, for example, as described herein). Preferably, the encoded sequence is at least 75%, more preferably at least 85%, and most preferably at least 95% identical at the amino acid level to the sequence of comparison. If nucleic acid sequences are directly compared, a “substantially identicar” nucleic acid sequence is one which is at least 85%, more preferably at least 90%, and most preferably at least 95% identical to the sequence of comparison. The invention encompasses polynucleotide sequences being 60-99% substantially identical to the nucleic acid sequences set forth is SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, or any combination thereof. The length of nucleic acid sequence comparison will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 100 nucleotides. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).

[0043] As used herein, “positionedfor expression” means that the nucleic acid molecule is operably linked to a sequence which directs transcription and translation of the nucleic acid molecule.

[0044] As used herein, “purified antibody” means an antibody which is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, antibody.

[0045] As used herein, “specifically binds” means an antibody which recognizes and binds a Conus protein disulfide isomerase, but which does not substantially recognize and bind other molecules in a sample (for example, a biological sample). An antibody which “specifically binds” such a polypeptide is sufficient to detect protein product in such a biological sample using one or more of the standard immunological techniques available to those in the art (for example, Western blotting or immunoprecipitation).

[0046] As used herein, “peptide,” “polypeptide” and “protein” include polymers oftwo or more amino acids of any length. No distinction, based on length, is intended between a peptide, a polypeptide or a protein.

[0047] As used herein, “protein disulfide isomerase activity” includes fragments of a protein disulfide isomerase which retain protein disulfide isomerase activity as assayed using methods known in the art or disclosed herein. Fragments of a protein disulfide isomerase, which retain protein disulfide isomerase activity, include N-terminal truncations, C-terminal truncations, amino acid substitutions, deletions and addition of amino acids (either internally or at either terminus of the protein).

[0048] Yeast cells have been used for expression of disulfide-rich polypeptides and co-expression of PDI has resulted in improved recombinant expression of properly folded disulfide-rich peptides (Kowalski et al., 1998; Shusta et al., 1998). The invention provides an important advance in this field of technology. For example, the identification of the Conus protein disulfide isomerase provides a simple and inexpensive means to increase the production of commercially important disulfide bond-containing proteins. Conus PDI provides a useful folding catalyst for production of properly folded conotoxins, for example, when produced in a recombinant system. Also, a Conus PDI is useful as a folding catalyst of conotoxins that are synthesized chemically and folded in vitro. Because the protein disulfide isomerase may be recombinantly expressed in combination with a commercial protein of interest or may be used as an isolated and purified reagent, the invention enables the enhancement of disulfide bond formation during in vivo commercial protein production or at subsequent in vitro purification steps, or both. Moreover, to further maximize disulfide bond formation, Conus protein disulfide isomerase proteins maybe used in conjunction with other disulfide bond-forming enzymes, for example, Erolp, or cell extracts, for example, in vitro translation systems such as rabbit reticulocyte lysates or wheat germ systems. Proper formation of disulfide bonds results in the production of batches of recombinant proteins exhibiting higher yields of properly folded products; this maximizes protein activity and minimizes the presence of inactive species and/or species which may be capable of triggering immunological side effects.

[0049] The invention relates to protein disulfide isomerases from Conus snails, to nucleic acid sequences encoding the Conus protein disulfide isomerases and to a method for using the nucleic acid or protein sequences for folding disulfide-containing proteins.

[0050] In one aspect, the invention relates to the amino acid sequence of C. textile protein disulfide isomerase. The amino acid sequences of C. textile protein disulfide isomerases are set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8. In another embodiment, the invention relates to a protein disulfide isomerase that has at least 95% identity with the amino acid sequence set forth in SEQ ID NO:2 and has protein disulfide isomerase activity. In another embodiment, the invention relates to a protein disulfide isomerase that has at least 95% identity with the amino acid sequence set forth in SEQ ID NO:4 and has protein disulfide isomerase activity. In another embodiment, the invention relates to a protein disulfide isomerase that has at least 95% identity with the amino acid sequence set forth in SEQ ID NO:6 and has protein disulfide isomerase activity. In another embodiment, the invention relates to a protein disulfide isomerase that has at least 95% identity with the amino acid sequence set forth in SEQ ID NO:8 and has protein disulfide isomerase activity. Protein disulfide isomerase activity can be assayed as described herein or by methods known in the art.

[0051] In another aspect, the invention relates to functional fragments of Conus protein disulfide isomerase. The PDI of SEQ ID NO:2 has two thioredoxin domains, amino acids 23-134 and 365-470, and a calsequestrin domain from amino acids 23 to 280. The PDI of the invention includes fragments ofthe PDI wherein protein disulfide isomerase activity is retained. For example, a peptide having a single thioredoxin domain, wherein the single domain has protein disulfide isomerase activity, is an aspect of the invention. The domain structure may be assayed, for example, using computer programs such as those described in Altschul, et al. (1997), Nucleic Acids Res. 25:3389-3402.

[0052] In another aspect, the invention relates to vectors containing the nucleic acid encoding a protein disulfide isomerase of the present invention. In one embodiment, the vector is an expression vector.

[0053] Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant protein. The precise host cell used is not critical to the invention. The host cells produce the protein disulfide isomerase when grown under suitable growth conditions. Suitable host cells include, but are not limited to, a eukaryotic host, such as insect cell lines (for example, HIGH FIVE™ from INVITROGEN™ (BTI-TN-5B1-4), derived from Trichoplusia ni egg cell homogenates), Sf9 or Sf21 cells, Lepidopteran insect cells, mammalian cell lines (for example, primary cell cultures or immortalized cell lines, such as COS 1, NIH3T3, HeLa, 293, CHO and U266), transgenic plants, plant cells, Drosophila Schneider2 (S2) cells, Baculovirus Expression Systems, Saccharomyces, Schizosaccharomyces, a prokaryotic host, such as Aspergillus, E. coli, Bacillus or the like. Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; INVITROGEN™; GIBCO™; see also, e.g., Ausubel et al., supra). The method of transformation or transfection and the choice of expression vehicle (vector) will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al., supra; expression vehicles may be chosen from those provided, for example, in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987) or known in the art.

[0054] A conotoxin or protein disulfide isomerase polypeptide is produced in a rnamalian system, for example, by a stably transfected mammalian cell line. A number of vectors suitable for stable transfection of mammalian cells are available; methods for constructing such cell lines are also publicly available, e.g., in Ausubel et al., supra. In one example, cDNA encoding the protein disulfide isomerase protein is cloned into an expression vector which includes the dihydrofolate reductase (DHFR) gene. Integration of the plasmid and, therefore, the PDI protein-encoding gene into the host cell chromosome is selected for by inclusion of 0.01-300 μM methotrexate in the cell culture medium (as described in Ausubel et al., supra). This dominant selection may be accomplished in most cell types. Recombinant protein expression may be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described in Ausubel et al., supra; such methods generally involve extended culture in medium containing gradually increasing levels of methotrexate. DHFR-containing expression vectors commonly used for this purpose include pCVSEII-DHFR and pAdD26SV(A) (described in Ausubel et al., supra). Any of the host cells described above or, preferably, a DHFR-deficient CHO cell line (for example, CHO DHFR cells, ATCC Accession No. CRL 9096) are among the host cells preferred for DHFR selection of a stably transfected cell line or DHFR-mediated gene amplification.

[0055] In another example, cDNA encoding the protein disulfide isomerase is cloned into a vector or an expression vector which includes a selectable marker gene. Methods for selecting cell lines containing the vector or expression vector are known in the art and described in Ausubel et al., supra.

[0056] In another aspect, the invention relates to host cells containing an expression cassette or expression vector with the protein disulfide isomerase encoding a nucleic acid of the invention and an expression cassette with a nucleic acid sequence encoding a disulfide-containing protein which is to be properly folded and expressed. Such proteins include conotoxins.

[0057] In another aspect, the invention relates to the use of a protein disulfide isomerase of the invention for the folding of disulfide-rich proteins.

[0058] In another aspect, the invention relates to an antibody that selectively binds to a PDI isolated from Conus, for example, an antibody that selectively-binds to a region of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.

[0059] In another aspect, the invention relates to nucleic acid derivatives and allelic variations of the nucleic acids and proteins disclosed in SEQ ID NO:1 through SEQ ID NO:8. The invention also relates to fragments of either the nucleic acid or protein which encodes or retains protein disulfide isomerase activity as assayed using methods known in the art or disclosed herein.

[0060] In another aspect, the invention relates to a method for increasing protein secretion of overexpressed gene products by enhancing protein disulfide isomerase activity within a cell. In eukaryotic cells, correct folding and assembly of a secreted polypeptide occurs in the endoplasmic reticulum (ER). (FIG. 1 illustrates the typical secretory/folding pathway and maturation process for a conopeptide.) Correct folding is a prerequisite for transport from the ER through the secretory pathway, with misfolded proteins being retained in the ER. Therefore, increased expression of the PDI of the invention is used to reduce the percentage of misfolded proteins and thereby increase secretion of the overexpressed gene product. At least one PDI of the invention is expressed in a host cell which overexpresses a gene product. “Overexpression,” as used herein, means a gene product that is expressed at levels greater than normal wild-type endogenous expression for that gene product. Thus, an overexpressed gene product is produced by, for example, introduction of a recombinant expression construct or altering endogenous expression levels, for example, by mutation or induction of transcription.

[0061] In another aspect, the invention is used to reconfigure human or animal hair. The compositions of the invention can be used for the treatment or degradation of scleroproteins, especially hair, skin and wool, dehairing and softening hides, treatment and cleaning of fabrics, as additives to detergents, thickening and gelation of food and fodder, strengthening of gluten in bakery or pastry products, and as pharmaceuticals for the alleviation of eye sufferings.

[0062] In another aspect, the PDI of the invention, having improved properties for a particular application, can be prepared by a variety of methods based on standard recombinant DNA technology, for example, by using site-directed or random mutagenesis to modify the genes encoding a Conus PDI to produce one or more amino acid changes, by inhibiting or otherwise avoiding dimerization of the subunits of PDI to provide PDI monomers, by producing partial monomers of PDI-lacking regions of the amino-terminus or carboxy-terminus of the PDI. Furthermore, the invention includes active truncated forms ofthe Conus PDIs which retain protein disulfide isomerase activity, wherein at least one redox site subunit (CGHC (SEQ ID NO:19)) is retained. For example, the minimal redox sites are located at amino acid positions 53-56 and 401-404 of SEQ ID NO:2.

[0063] In another aspect, the invention relates to a nucleic acid encoding an active recombinant protein disulfide isomerase of the invention. The nucleic acid may comprise introns and/or regulatory elements native to the Conus PDI. Alternatively, the nucleic acid may comprise introns and/or regulatory elements derived from other organisms or synthetic constructs.

[0064] A nucleic acid or fragment thereof has substantial identity with another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70% of the nucleotide bases, usually at least about 80%, more usually at least about 85%, and more preferably at least about 90% of the nucleotide bases. A protein or fragment thereof has substantial identity with another if, when optimally aligned, there is an amino acid sequence identity of at least about 70% identity with an entire naturally occurring protein or a portion thereof, usually at least about 80% identity, preferably at least about 85% identity, preferably at least about 90% identity, and more preferably at least about 95-98% identity.

[0065] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis., 53705, or BLAST software available from the National Library of Medicine). Examples of useful software include the programs, PILE-UP™ and PRETTYBOX™. Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0066] “Identity” means the degree of sequence relatedness between two polypeptide or two polynucleotides sequences as determined by the identity of the match between two strings of such sequences, such as the full and complete sequence. Identity can be readily calculated. While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991). Methods commonly employed to determine identity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipman, D., SIAM J. Applied Math, 48:1073 (1988). Preferred methods to determine identity are designed to give the largest match between the two sequences tested. Such methods are codified in computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, GCG (Genetics Computer Group, Madison Wis.) program package (Devereux, J., et al., Nucleic Acids Research 12(1), 387 (1984)), BLASTP, BLASTN, FASTA (Altschul et al. (1990); Altschul et al. (1997)). The well-known Smith Waterman algorithm may also be used to determine identity.

[0067] As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% “identity” to a reference nucleotide sequence, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides ofthe reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations relative to the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0068] Alternatively, substantial homology or (similarity) exists when a nucleic acid or fragment thereof will hybridize to another nucleic acid (or a complementary strand thereof) under selective hybridization conditions. Selectivity of hybridization exists when hybridization which is substantially more selective than total lack of specificity occurs. Typically, selective hybridization will occur when there is at least about 55% homology over a stretch of at least about nine nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. The length of homology comparison, as described, may be over longer stretches and, in certain embodiments, will often be over a stretch of at least about 14 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.

[0069] Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, the length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of 30° C., typically in excess of 37° C., and preferably in excess of 45° C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. The stringency conditions are dependent on the length of the nucleic acid and the base composition of the nucleic acid, and can be determined by techniques well known in the art, for example, Ausubel, 1992; Wetmur and Davidson, 1968.

[0070] Thus, as herein used, the term “stringent conditions” means hybridization will occur only if there is at least 80%, preferably at least 90%, more preferable at least 95% and most preferably at least 97% identity between the sequences. Such hybridization techniques are well known to those of skill in the art. Stringent hybridization conditions are as defined above or, alternatively, conditions under overnight incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

[0071] Hybridization techniques and procedures are well known to those skilled in the art and are described, for example, in Ausubel et al., supra, and Guide to Molecular Cloning Techniques, supra. If desired, a combination of different oligonucleotide probes may be used for the screening of the recombinant DNA library. The oligonucleotides are, for example, labeled with 32P using methods known in the art, and the detectably labeled oligonucleotides are used to probe filter replicas from a recombinant DNA library. Recombinant DNA libraries (for example, Conus cDNA libraries) may be prepared according to methods well known in the art, for example, as described in Ausubel et al., supra. Such libraries may be generated using standard techniques.

[0072] An alignment of PDIs from Conus textile (Ct-PDI) (SEQ ID NO:2), silkworm (SEQ ID NO:9), sea urchin (SEQ ID NO:10), rat (SEQ ID NO:11), human (SEQ ID NO:12), Drosophila (Fly-PDI) (SEQ ID NO:13), and C. elegans (SEQ ID NO:14) is illustrated in FIG. 2. This figure indicates the nonvariant amino acids by the presence of a * under that amino acid position.

[0073] An alignment of four Conus textile Protein Disulfide Isomerases ofthe invention is illustrated in FIG. 3. The four sequences shown correspond to Conus textile PDI (SEQ ID NO:2) and three isoforms, texl (SEQ ID NO:4), tex2 (SEQ ID NO:6) and tex3 (SEQ ID NO:8). The isolated PDIs from Conus textile demonstrate a high degree of sequence identity relative to the interspecies sequence identity.

[0074] Large amounts of the nucleic acids of the invention may be produced by (a) replication in a suitable host or transgenic animal or (b) chemical synthesis using techniques well known in the art. Constructs prepared for introduction into a prokaryotic or eukaryotic host may comprise a replication system recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment. The choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, a viral or phage vector, a transposable element, an integrating vector or an extrachromosomal element, such as a minichromosome or an artificial chromosome. Such vectors may be prepared by means of standard recombinant techniques well known in the art. See for example, see Ausubel (1992); Sambrook and Russell (2001); and U.S. Pat. No. 5,837,492.

[0075] Large amounts of the protein ofthe invention may be produced (a) by expression in a suitable host or transgenic animal, (b) in vitro, for example, using a T7 system (see, for example, Ausubel et aL, supra, or other standard techniques) or (c) by chemical synthesis using techniques well known in the art (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, such as a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Secretion signals may also be included where appropriate which allow the protein to cross and/or lodge in cell membranes. Other signals may also be included where appropriate which allow translocation to a specific cellular compartment (for example, endoplasmic reticulum, nucleus, peroxisome, etc.) and/or retention in a compartment. For example, the amino acid KDEL (SEQ ID NO:20) can be used to retain proteins in the endoplasmic reticulum. Such vectors may be prepared by means of standard recombinant techniques well known in the art. See for example, see Ausubel (1992); Sambrook and Russell (2001); and U.S. Pat. No. 5,837,492.

[0076] Once the recombinant protein of the invention is expressed, it may be isolated, for example, using affinity chromatography. In one example, an anti-Conus PDI protein antibody (for example, produced as described herein) may be attached to a column and used to isolate the PDI protein. Lysis and fractionation of PDI protein-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra).

[0077] Once isolated, the recombinant protein can, if desired, be further purified, for example, by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology, eds., Work and Burdon Elsevier, 1980).

[0078] The proteins of the invention may be cotranslationally, post-translationally or spontaneously modified, for example, by acetylation, famesylation, glycosylation, myristoylation, methylation, prenylation, phosphorylation, palrnitoylation, sulfation, ubiquitination and the like. See, Wold, F. (1981), Annu. Rev. Biochem. 50:783-814.

[0079] The protein disulfide isomerase of the invention is isolated following expression in a suitable host or chemical synthesis using techniques well known in the art. The isolated protein disulfide isomerase of the invention is used to correctly fold disulfide-containing proteins. The protein disulfide isomerase is contacted with the unfolded or misfolded protein and allowed to direct the proper folding of the protein. The correctly folded protein is isolated and purified using techniques well known in the art.

[0080] The nucleic acid encoding a protein disulfide isomerase of the invention is used to correctly fold disulfide-containing proteins in vivo using techniques well known in the art. In one embodiment, a suitable host is prepared which contains an expression vector containing a protein disulfide isomerase encoding nucleic acid of the invention and an expression vector containing a nucleic acid encoding a disulfide-containing protein. Such disulfide-containing proteins include conotoxins. Nucleic acids encoding conotoxins are well known in the art. See, U.S. Pat. No. 5,739,276. Nucleic acids encoding other disulfide-containing proteins are also well known in the art. In a second embodiment, a suitable host is prepared which contains an expression vector containing a protein disulfide isomerase encoding nucleic acid and a nucleic acid encoding a disulfide-containing protein. In either embodiment, the host cells are grown under conditions suitable for growth and expression of the protein disulfide isomerase and the disulfide-containing protein. The protein disulfide isomerase acts on the disulfide-containing protein in vivo to properly fold the protein.

[0081] The protein disulfide isomerase is cloned into an expression cassette, which is driven by a promoter appropriate for the host cell and contains other transcriptional and translational signals necessary for expression of the PDI in the host cell. The protein disulfide isomerase is expressed in mammalian cells using standard techniques known in the art. For example, the PDI is placed under the control of a promoter, such as the Drosophila inducible metallothionein promoter, and introduced into Drosophila cells. The PDI is followed by a poly (A) signal recognized by the host cell.

[0082] The protein disulfide isomerase of the invention can be expressed as a fusion protein, wherein the PDI gene is fused in frame to a disulfide-rich peptide. The fusion may include additional sequences between the PDI gene and the disulfide-rich peptide, for example, a proteolytic cleavage site. The fusion may also include a signal sequence, ER retention signals, and the like.

[0083] Anti-PDI Antibodies:

[0084] Using the PDI polypeptide described herein or isolated as described above, anti-PDI antibodies may be produced by any standard technique. In one particular example, a PDI cDNA or cDNA fragment encoding a conserved PDI domain is fused to GST, and the fusion protein produced in E. coli by standard techniques. The fusion protein is purified on a glutathione column, also by standard techniques, and is used to immunize rabbits. The antisera obtained is then itself purified on a GST-PDI affinity column and is shown to specifically identify GST-PDI, for example, by Western blotting.

[0085] Polypeptides for antibody production may be produced by recombinant or peptide synthetic techniques (see, e.g., Solid Phase Peptide Synthesis, supra; Ausubel et al., supra).

[0086] For polyclonal antisera, the peptides may, if desired, be coupled to a carrier protein, such as KLH as described in Ausubel et al., supra. The KLH peptide is mixed with Freund's adjuvant and injected into guinea pigs, rats, goats or, preferably, rabbits. Antibodies maybe purified by any method of peptide antigen affinity chromatography.

[0087] Alternatively, monoclonal antibodies may be prepared using a PDI polypeptide (or immunogenic fragment or analog) and standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292,1976; Hammerling et al., In: Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981; Ausubel et al., supra).

[0088] In addition antibody fragments which contain specific binding sites for Conus PDIs may be generated. For example, such fragments include, but are not limited to, the F(ab′)2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W. D. et al. (1989), Science 256:1275-1281).

[0089] Once produced, the polyclonal or monoclonal antibody is tested for specific PDI recognition by Western blot or immunoprecipitation analysis (by the methods described in Ausubel et al., supra). Antibodies which specifically recognize a PDI polypeptide described herein are considered to be useful in the invention.

[0090] The invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.

EXAMPLE I

Purification of Protein Disulfide Isomerase from Conus textile

[0091] Preparation and purification of protein disulfide isomerase from Conus textile was performed as described by Lambert and Freedman (Lambert and Freedman, 1983). Dry venom ducts were ground under liquid N2 and then homogenized in 10 mM Tris.HCl (pH 7.8) containing 0.25 M sucrose and 5 mM EDTA. The nuclei, whole cells, mitochondria and lysosomes were removed by centrifugation. The supernatant was adjusted to pH 5.2 and stirred to precipitate the microsomal fraction. The microsomal material was harvested by centrifugation and the pellet was homogenized in 10 mM Tris.HCl (pH 7.8), stirred for 2 hours and then centrifuged. The soluble microsomal fraction was dialyzed against the same buffer for 48 hours.

[0092] (NH4)2SO4 was added to the dialyzed extract to 50% saturation. After stirring for 1 hour, the solution was centrifuged and the pellet was discarded. Additional (NH4)2SO4 was added to the supernatant to the final concentration of 85% saturation and the solution was centrifuged as before. The pellet was dissolved in 0.1 M Tris.HCl (pH 7.8) and dialyzed against the same buffer for 24 hours.

[0093] The dialyzed material was loaded onto a DEAE-Sephadex A-50 column, equilibrated with 0.1 M Tris-HCl (pH 7.8). For elution, a linear NaCl gradient was applied. PDI is detected between 0.3 and 0.4 M NaCl by SDS-polyacrylamide gel electrophoresis.

[0094] Edman Sequencing of PDI from Conus

[0095] The venom duct was dissected from Conus textile and immediately divided into four equal parts, FIG. 4A. Each part of the venom duct was grounded under liquid nitrogen. Extraction was performed in 1 ml of 20% acetonitrile, 0.1% TFA at 4° C. After 1 hour of incubation, the solution was centrifuged and the resulted pellet was dissolved in 1 ml of 10% acetonitrile, 0.1% TFA. The solution was mixed for 1 hour at 4° C. and then centrifuged. 50 μl of supernatant was lyophilized and dissolved in 30 μl of SDS-electrophoresis buffer, boiled for 5 minutes and applied on 4-20% Tris-glycine gel. The proteins were then electroblotted onto an Immobilion PVDF membrane (0.45 μm) (Millipore) for 1 hour at 50 V. Proteins were visualized using Coomassie Blue staining and the protein band of 55 kDa was cut out from the membrane. Amino acid sequencing was performed by the Edman degradation method.

[0096] From the Coomassie-stained gel, shown in FIG. 4B, it is apparent that a band corresponding to 55 kDa protein was predominant and was found in each section of the venom duct. This band had a similar molecular weight to that of a bovine PDI (used as a reference in lane 5). The following sequence was obtained from 10 cycles: EEVEQEENVY (SEQ IDNO:21). This sequence matched the predicted amino acid sequence ofthe mature protein disulfide isomerase cloned from a venom duct of Conus textile (SEQ ID NO:2), confirming that the 55 kDaband corresponds to PDI. It thus appears that PDI is a major protein component of venom ducts. The Conus PDI was also not cross-reactive with polyclonal antibodies against bovine PDI (data not shown), suggesting substantial differences between bovine and Conus PDI.

EXAMPLE II

In vitro Folding of α-GI and Pro-GI with Protein Disulfide Isomerase

[0097] Synthesis and Folding of Reference Pro-GI

[0098] Pro-GI was chemically synthesized using two different types-of-thiol protecting groups. Cys1 and Cys3 were blocked with trityl (Trt) groups, and Cys2 and Cys4 were blocked with the acetamidomethyl (Acm) groups. The peptide was cleaved from the resin concurrent with the removal of the Trt protecting groups, and the first disulfide bond was formed in 0.1 M Tris.HCl (pH 8.7) containing 1 mM EDTA, 1 mM oxidized glutathione (GSSG) and 2 mM reduced glutathione (GSH) at 20 μM peptide concentration. After 1 hour of oxidation at room temperature, the reaction was quenched with 8% formic acid. The pro-GI concentration was determined spectrophotometrically using the molar absorbance coefficient at 274.5 nm, ε=1420 M−1×cm−1 (Pace et al., 1995). Pro-GI with the first disulfide bridge oxidized (Cys1-Cys3) was purified on a Vydac C18 semipreparative HPLC column to approximately 90% purity. The peptide was eluted from the column using a two-buffer system in a linear gradient of 10% solvent B to 30% solvent B over 60 minutes where solvent A is 0.1% trifluoroacetic acid (TFA) and solvent B is 90% acetonitrile with 0.1% TFA. The Acm protecting groups were removed from the remaining two cysteines (Cys2 and Cys4) and the cysteines were oxidized in a single step using iodine oxidation. The correctly folded peptide was purified as described before to over 90% purity. This material was used as a reference to follow the PDI catalyzed folding of pro-GI.

[0099] In general, this approach involves the use of purified PDI in combination with any in vitro refolding reaction. In another example, a recombinant protein of interest is expressed (for example, in an E. coli or mammalian cell culture system) and is treated with a denaturant, such as guanidine hydrochloride. The protein preparation is then allowed to refold by dilution of the denaturant, and proper disulfide bond formation is promoted during this renaturation step by the presence of PDI protein in the reaction mixture. If desired, the PDI protein may be added in a buffer combined with oxidized and reduced glutathione and/or other purified PDIs or chaperones. Additional proteins may be added, such as Ero1. The PDI may also be added to in vitro transcription and/or translation systems such that improved folding is achieved.

[0100] Identification of Native proGI and proPVIA:

[0101] Identification ofproGI folding species, containing the native disulfides, was verified by coelution with correctly folded proGI, which was produced in the two-step folding reaction. Identification of proPVIA folding species containing the native disulfides was based on limited digestion by bovine trypsin. The proPVIA forms generated by oxidation in the presence of glutathione were dissolved in a solution of bovine trypsin (150 ng/ml of 0.1 M Tris/HCl, pH 8.7) to give an enzyme/substrate weight ratio of 1:100. After 16 hours of incubation at room temperature the digestion products were separated from one another on a Vydac C18 analytical HPLC column. Solvents A (0.1% trifluoroacetic acid) and B (90% acetonitrile and 0.1% trifluoroacetic acid) were mixed to form a linear gradient of 15% to 60% over the course of 30 minutes. The form of proPVIA with the retention time corresponded to the δ-PVIA, containing native disulfides, was isolated and coeluted with native material. The HPLC coelution of the isolated digestion product with the native peptide confirmed the identity of this species as having a native configuration. Additionally, the identity of digestion product, corresponding to the native δ-PVIA, was confirmed by electrospray mass spectrometry (ESI-MS).

[0102] Mass Spectrometry:

[0103] Electrospray mass spectrometry of peptides used in this study was performed with Quatro II Micromass mass spectrometer and Masslynx software. Samples were dissolved in methanol/water (1:1, v/v) containing 0.01% TFA. Molecular masses of all peptides were within 1.0 atomic unit from those expected from the amino acid sequence.

[0104] Oxidative Folding Reactions:

[0105] Standard oxidative folding reactions of α-GI and proGI were performed in 0.1 M Tris/HCl, pH 8.7, containing 1 mM EDTA, 0.5 mM GSSG and 5 mM GSH, at 22° C. The folding experiments catalyzed by bovine protein disulfide isomerase (PDI) were carried out in 0.1 M Tris/HCl, pH 7.5, containing 1 mM EDTA, 0.1 mM GSSG and 2 μM PDI, at 0° C. The reaction was initiated by adding the linear peptide to the folding mixture to the final concentration of 20 μM. After an appropriate time, the reaction was quenched by adding formic acid to the final concentration of 8%. The disulfide-bounded species were separated on a Vydac C18 analytical HPLC column. Solvents A (0.1% trifluoroacetic acid) and B (90% acetonitrile and 0.1% trifluoroacetic acid) were mixed to form a linear gradient of 5% to 30% for 40 minutes and 15% to 25% for 60 minutes for elution of α-GI and proGI, respectively.

[0106] Standard oxidative folding reactions of δ-PVIA and proPVIA were performed in 0.1 M Tris/HCl, pH 7.5, containing 1 mM EDTA, 1 mM GSSG and 2 mM GSH, at 0° C. and a peptide concentration of 10 μM. After 16 hours the reaction was quenched as described before. The disulfide-bounded species were separated on a Vydac C18 analytical HPLC column. Solvents A and B were mixed to form a linear gradient of 15% to 60% for 30 minutes for both δ-PVIA and proPVIA.

[0107] Concentrations of all peptides were determined spectrophotometrically using the molar absorbance coefficient at 274.5 nm, ε=1420 M−1×cm−1 for α-GI and proGI or ε=M−1×cm−1 and ε=M−1×cm−1 for δ-PVIA and proPVIA, respectively (Pace et al., 1995).

[0108] Synthesis of Precursors for α-GI and δ-PVIA Conotoxins:

[0109] Two model disulfide-rich peptides were chemically synthesized, denoted as proGI and proPVIA, for the respective conotoxins, α-GI and δ-PVIA. The sequences of synthetic peptides were identical with the native ones, predicted from cDNA sequence analysis.

[0110] The proGI was synthesized in two versions, where either all four cysteines were Trt-protected, or two (Cys1 and Cys3) were protected by Trt groups and two (Cys2 and Cys4) by Acm groups. The first variant of proGI, all four cysteines Trt-protected, was used in this experiment. The thermodynamic and kinetic parameters of folding were compared with the same parameters for the mature toxin. The proGI with native disulfides, folded according to the two-step folding procedure, was used as an HPLC reference for the coelution studies.

[0111] The proPVIA was synthesized with all six cysteine residues protected by Trt groups. After cleavage from the resin and purification on HPLC reverse phase column, the proPVIA was used in the folding experiments. Since a reference with native cysteine connectivity for proPVIA folded was not available, an HPLC reference was generated through trypsin digestion of the native form of proPVIA. Coelution experiment with digestion product of proPVIA and native δ-PVIA confirmed correct cysteine connectivity of folded proPVIA. Additionally, we measured the molecular weight of proPVIA folded digestion product. Molecular mass was within 0.5 atomic mass unit from those determined and calculated for native δ-PVIA.

[0112] Synthetic precursors were purified to over 90% homogeneity on reverse-phase C18 HPLC column and the identity was confirmed by electrospray ionization mass spectrometry (Table 1).

2TABLE 1Mass of synthetic precursors.Synthetic PrecursorMasspro-GI4562.1 Dapro-PVIA6476.0 Da

[0113] Role of Propeptide in Uncatalyzed and PDI-Catalyzed Folding:

[0114] Since the propeptide sequence may stabilize not only native conformation, but also the conformation(s) favoring productive folding pathway(s), we assessed if the propeptide can change the rate of the oxidative folding of a conotoxin peptide. The redox buffer was used to favor a steady-state accumulation of many different folding species. δ-PVIA accumulated to only 1-3% in the presence of 1 mM GSSG/2 mM GSH and even minor changes in the thermodynamic stability of the native conformation would be easily detected. Under our in vitro folding conditions, the propeptide affected neither kinetics nor thermodynamics for forming the native disulfide bonds.

[0115] Oxidative folding of α-GI, pro-GI, δ-PVIA and pro-PVIA was carried out in buffered solutions containing oxidized and reduced glutathione, as described above. After an appropriate time, the folding reactions were quenched by acidification and analyzed by reversed-phase HPLC. The steady-state distribution of the folding species for pro-GI, pro-PVIA and the corresponding mature species α-GI and δ-PVIA are shown in FIGS. 5A-5D. The equilibrium accumulation of the native forms was similar for the mature toxins and the respective propeptide-containing variants: 20% yield of the native α-GI, as compared to 27% yield ofthe native pro-GI. Despite no effects of propeptide on a relative accumulation of the native forms, the number of the steady-state folding species was significantly reduced for pro-GI, as compared to that for α-GI (FIGS. 5A and 5C). There was no significant difference in accumulation of the native form of δ-PVIA compared with pro-PVIA: 2.1% versus 3.3%, as well as in the number of accumulated folding species.

[0116] To explore a possibility that propeptide could influence the oxidative folding under different experimental conditions, we screened several factors, such as temperature, redox potential, denaturants or osmolytes. As summarized in FIG. 6, varying the folding environment in the presence of the N-terminal propeptide sequence did not significantly change the steady-state distribution ofthe native species. The accumulations of the native α-GI and pro-GI were equally sensitive to the different conditions, despite changes in the temperature from 0° C. to 37° C., redox potential from 1:1 to 1:10 GSSG/GSH or the presence of other folding additives, such as urea, glycerol, nonionic detergents or organic cosolvents. In the case of 8-PVIA and pro-PVIA, addition of the nonionic detergent Tween-40 increased the accumulation of the native forms by 3- and 1.7-fold, respectively. These results confirmed that the propeptide sequences did not directly participate in the stabilization of the native conotoxins.

[0117] We also investigated whether the propeptide could influence the folding rates. The kinetics for forming a first disulfide bond is represented by a rate of disappearance of a linear form. These rates were determined under two different experimental conditions: (i) in the presence of 0.5 mM GSSG and 5 mM GSH, where the folding rates are determined by intramolecular rearrangement steps and (ii) with 0.1 mM GSSG, where folding rates are determined by the reactivity of the peptide Cys thiols (reference Creighton, Goldenberg). As illustrated in FIGS. 7A-D, 8A and 9, the disappearance of the linear forms was comparable for both α-GI (kapp=0.039 min-1 at 0.1 mM GSSG) and pro-GI (kapp=0.039 min-1 at 0.1 mM GSSG). In the presence of 0.5 mM GSSH and 5 mM GSH, the apparent rates for α-GI and pro-GI were also similar: kapp=0.084 min-1 and kapp=0.073 min-1, respectively (FIG. 6). Thus, the propeptide sequence did not affect the early folding steps. In addition, the formation of the native α-GI or pro-GI and the appearance of the other folding species were almost identical for the mature toxin and propeptide-containing variant, suggesting that the propeptide did not change the overall folding rates, FIGS. 7A-D, 8A and 9. As will be apparent to a person of skill in the art, the methods and results of this experiment are applicable to other proteins.

[0118] Comparison of the rates of the first disulfide bond formation in the fully reduced α-GI and proGI shows that the disappearance of both linear forms is comparable and propeptide does not affect early steps of the oxidative folding of conotoxins (FIGS. 7A-D, 8A and 9). Also formation of the native forms and appearance of the other folding species did not differ significantly between mature toxin (α-GI) and the propeptide-containing variant (proGI) (FIGS. 7A-7D, 8A and 9). The results demonstrate that the propeptide did not change the rate of oxidative folding.

[0119] As previously decribed, efficient disulfide bond formation in eukaryotic cells is assisted by protein disulfide isomerase. To determine the catalytic effect of PDI on the propeptide-facilitated folding, we carried out the α-GI and proGI oxidation experiments in the presence of bovine PDI (FIGS. 7B, 7D, 8A and 9). The reactions were performed at an enzyme-to-substrate molar ratio of 1:10 and under cysteine residue reactivity, i.e., under very low concentration of GSSG, 0.1 mM.

[0120]
FIGS. 7B, 7D, 8A and 9 show the kinetics ofthe PDI-catalyzed folding of α-GI and proGI (for example, FIGS. 7B and 7D, respectively) compared with uncatalyzed reactions (FIGS. 7A and 7C). The folding reactions were carried out under identical experimental conditions as described in the previous section, except that PDI was added to the folding mixture prior to the addition of the linear peptide. Under folding conditions with a mixture of 0.5 mM GSSG and 5 mM GSH, PDI was primarily present in the reduced form (estimated PDI(red)/PDItotal was at least 97%, based on Kox of 1.3 mM (pH 7.4)—Darby, Creighton 1995, or at least 99% based on Kox of 0.7 mM (pH 8.0) Schwaller, Wilkinson, Gilbert 2003). In the presence of 0.1 mM GSSG, the enzyme was predominantly in the oxidized state.

[0121] To determine the step ofthe toxin folding pathway catalyzed by PDI, we compared both the disappearance of linear forms, formation of native species and appearance of other folding species. Addition of PDI led to a significantly higher accumulation of the native formi of both α-GI and proGI. The significantly faster disappearance of the linear form was accompanied by the faster accumulation of the native form. The rates for forming a first disulfide (disappearance of the linear form) were identical for α-GI and proGI, kapp=0.039 min-1 (α-GI) and kapp=0.039 min-1 (proGI). However, there were significant differences in the kinetics of accumulation of the native form between mature and precursor toxin. As shown in FIG. 9D, the presence of PDI had a dramatic effect on the accumulation of the native form of proGI, compared to that of α-GI (FIG. 9B) in the early steps of folding reaction. After the first five minutes in the presence of PDI, the amount of the native form of proGI increased 10.8-fold, whereas the amount of the native form of α-GI increased only 3.4-fold. Similarly, after the next five minutes, the effect of PDI catalysis is about 3 times higher for the propeptide-containing form (FIG. 8A). The single-exponential fit to the experimental points yielded kapp=0.046 min1 (α-GI) and kapp=0.145 mind (proGI). This 3-fold increase in the folding rates is further illustrated in FIG. 8B in a form of half-times. It is apparent that the PDI-catalyzed formation of the native pro-GI is more efficient, as compared to that of the mature conotoxin.

[0122] The rates of the PDI-catalyzed formation of the first disulfide bond in α-GI and proGI, measured by the time dependence of the disappearance of the linear form, were found to be very comparable (compare FIGS. 7B and 7D). However, in the case of α-GI, the disappearance of the fully reduced form is related to production of nonnative folding species. There was significantly lower accumulation of the nonnative folding species for proGI (FIGS. 7A-7D, 8A and 9A-9D). After ten minutes of folding, an accumulation of 44.8% of nonnative proGI folding species was observed, comparable with uncatalyzed formation of nonnative proGI folding species (46.3%). At the same time point, we observed almost 70% nonnative folding species in the PDI-catalyzed reaction of α-GI, over 1.5 times more when compared with uncatalyzed reaction (FIGS. 7A and 7B). These differences were not observed for the uncatalyzed folding (FIGS. 7A and 7C), suggesting that PDI is more efficient in rearrangement of the precursor folding species, as compared to that of the mature peptide. During the later time points, the accumulation of the other folding species was lower for α-GI, consistent with the equilibration experiments (the mature α-GI was more stable under a strongly oxidizing environment, as compared to proGI). Therefore, PDI did not change the thermodynamic stability of conotoxins, but rather influenced the productive folding pathway(s). Thus, PDI increased the rate of disulfide bond isomeration in the α-GI form and the rate and extent of natively folded proGI.

[0123] Interestingly, we did not observe such differences in the PDI-catalyzed folding of αGI and proGI when the reactions were carried out in the presence of 0.5 mM GSSG and 5 mM GSH. The enzyme increased the overall folding rates for both α-GI and pro-GI by approximately two-fold. The disappearance of the linear forms and the formation of the native forms for the precursor and the mature conotoxin were comparable (kinetic data not shown). The transient accumulation of the folding species was only slightly higher for α-GI in the presence of PDI (relative to the uncatalyzed reaction). Insufficient HPLC separation of the pro-GI folding intermediates precluded similar comparison for the precursor. The effect of the oxidative folding buffer, or redox system, on PDI-catalyzed folding of αGI and proGI results from changes in the catalytic efficiency of the enzyme over a range of reduced and oxidized glutathione concentrations (Lyles and Gilbert, 1991, Shwaller, Gilbert 2003). This effect was accounted for by the PDI redox state (the reduced dithiol form of PDI was required for the efficient folding of scrambled RNase), as well as by the redox state of the substrate itself.

[0124] It is apparent from the FIG. 2B in Lyles and Gilbert, 1991, that at the fixed 0.5 mM concentration of GSSG, increasing concentrations of GSH above 5 mM resulted in diminishing the differences between the PDI-catalyzed and uncaialyzed folding of RNase A. Indeed, we observed the identical phenomenon with the PDI-catalyzed and uncatalyzed folding of α-GI and pro-GI at 0.5 mM GSSH and 5 mM GSH.

[0125] This demonstrates that the propeptide alone does not appreciably contribute to the stability and the rate of oxidative folding of mature conotoxins. Further, bovine PDI facilitates the rate and amount of properly folded protein (FIGS. 7A-7D). The comparison of the folding species distribution at the early time points for α-GI and proGI, uncatalyzed versus PDI-catalyzed folding reaction, suggests that the propeptide could improve oxidative folding of conotoxins by making folding intermediate(s) better substrate(s) for PDI.

[0126]
FIG. 10 shows the activity of Conus PDI in the folding of αGI. Conus PDI increased the rate of disulfide bond isomeration and extent of natively folded αGI.

EXAMPLE III

Cloning of Conus textile Protein Disulfide Isomerase cDNA

[0127] Full-length C. textile PDI cDNAs were isolated by reverse transcription-PCR (RT-PCR) of venom duct RNA, using primers based on the highly conserved thioredoxin-like active site motif found in PDI genes isolated from other organisms. A typical Class 1 PDI gene would be expected to contain two nearly identical repeats ofthis sequence motif, separated by 750-1000 bp of intervening sequence. A forward PCR primer was designed to match the N-terminal region sequence of the thioredoxin site (amino acid sequence VEFYAPW (SEQ ID NO:15); primer PDIfor1: GTN GAR TTY TAY GCN CCN TGG (SEQ ID NO:16)) and a reverse primer (amino acid sequence WCGHCKQ (SEQ ID NO:17); primer PDIrev1: YTG YTT RCA RTG NCC RCA CCA (SEQ ID NO:18)) were designed to the C-terminal portion of the thioredoxin site. These PCR primers, based on the protein sequences of PDI enzymes from a variety ofdiffering organisms, contained degenerate codon usage to account for DNA sequence variation in the corresponding Conus genes. Venom duct tissue was dissected from C. textile snails and used to prepare mRNA and reverse-transcribed cDNA according to standard techniques. This venom duct cDNA was used for PCR amplification with the PDIfor1 and PDfrev1 primer pair. PCR amplification using a variety of different reaction conditions, thermostable polymerases, and cycling protocols consistently gave a predominant PCR product of ˜1000 bp, as well as a variety of minor products. The prominent ˜1000 bp PCR product was gel-purified and cloned into a plasmid vector, and several cloned isolates were sequenced. The DNA sequence of the cloned PCR product contained a single long open reading frame with significant homology to Class 1 PDI genes from other organisms (i.e., ˜55% identity to human PDI proteins) and confirmed that this PCR product represented a C. textile PDI isoform. As predicted, this PCR-generated cDNA represented the gene sequence extending between the two thioredoxin-like domains and lacked the 5′ and 3′ regions of the full-length cDNA. The DNA sequence of this initial PCR product was used to design nested PCR primers for 5′ and 3′ RACE procedures (rapid amplification of cDNA ends) to isolate the full-length cDNA. C. Textile venom duct cDNA was synthesized with 5′ and 3′ RACE adapters and used for RACE amplifications. The nested 5′ RACE primers generated a specific product of ˜350 bp, and the 3′ RACE primers gave a specific product of 1250 bp. These 5′ and 3′ RACE products were gel-purified, cloned into a plasmid vector, and sequenced. The sequences of each ofthese RACE products overlapped with the previously isolated central portion of the C. textile PDI cDNA, and together these 3 PCR-generated cDNAs could be merged to give the full-length cDNA sequence encoding the complete PDI protein. The full-length cDNA sequence contains a single long open reading frame encoding a C. textile Class 1 PDI protein of 502 amino acids. The cDNA sequence includes between about 30 and 140 bp of 5′ untranslated sequences and between about 65 and 850 bp of 3′ untranslated region sequences. Translation of the PDI ORF shown in SEQ ID NOs:1 and 5 initiates at the first ATG start codon from the 5′ end. Translation of the PDI ORF shown in SEQ ID NO:3 initiates at the third ATG of the 5′ sequence. The encoded proteins contain two thioredoxin-like domains, separated by ˜350 amino acids; each of these domains contains a cysteine redox-active site (—CGHC—) SEQ ID NO:19. The C. textile PDI enzyme contains a C-terminal ER retention signal, as predicted for a Class 1 enzyme functioning in the secretory pathway. The 3′ RACE clone terminates in a typical poly-A tail, preceded by a poly-A addition signal, indicating that this clone represents the true 3′ end of the mRNA. This initial cDNA sequence was generated by PCR using Taq polymerase and nested PCR amplifications utilizing up to 60 amplification cycles. It is possible that Taq polymerase misincorporation errors could be present in the initial sequence. To generate a cDNA clone of the entire coding region devoid of sequence errors, PCR primers were designed in the 5′ and 3′ untranslated regions immediately surrounding the open reading frame and used to amplify the complete 1500 bp coding region using an LA-PCR (long-accurate) protocol, proof-reading polymerase mixture, and only 20 amplification cycles. Amplification of C. textile venom duct cDNA gave a single, robust product at the predicted 1500 bp size. This product was cloned into a plasmid vector and completely sequenced on both strands to give the complete C. textile PDI nucleic acid sequence presented in SEQ ID NO:1.

[0128] In addition, we have isolated cDNA clones for three closely related PDI variants from C. textile venom duct. Although these sequences are distinct from any previously isolated PDI enzyme, they do display features typical of Class 1 PDI enzymes. In particular, the Conus enzymes have two conserved active site domains separated by ˜350 amino acids and a C-terminal endoplasmic reticulum retention signal.

EXAMPLE IV

Expression of Protein Disulfide Isomerase in Host Cells

[0129] PDI may also be used to catalyze proper disulfide bond formation in any in vivo protein expression system. By this approach, a full-length PDI-expressing nucleic acid is introduced into a host cell which also expresses a disulfide-rich protein of interest. Preferably, the PDI gene encodes a Conus PDI, and the disulfide-rich protein is a conotoxin, preferably, produced at high levels in the cultured cells. Any appropriate eukaryotic cell may be used for protein expression in conjunction with a PDI product. This technique may be used for the production of any protein which is disulfide-rich.

EXAMPLE V

Synthesis of Conus PDI in Host Cells

[0130] Protein disulfide isomerase (PDI) enzymes, characterized by the presence of highly conserved thioredoxin-like active site domains, catalyze the formation of cysteine-cysteine disulfide linkages in nascent proteins. The PDI activity isolated from Conus venom duct is demonstrated to facilitate the folding of disulfide-rich peptides, such as conopeptide precursors.

[0131] The quantity of PDI enzyme that can be isolated from venom duct is limited by the impracticality of obtaining large quantities of this tissue. Functional expression ofthe cloned Conus PDI enzymes in heterologous expression systems will allow production of large amounts of the enzyme. The recombinantly produced enzyme can be purified and used to facilitate folding of synthetic conopeptide precursors in an in vitro folding reaction. In addition, any cloned cysteine-containing conopeptide precursor gene can be co-expressed in a recombinant cell line that has been engineered to express high levels of the Conus PDI enzyme. The high levels of PDI enzymatic activity will promote efficient folding of the processed, bioactive conopeptide and allow higher yields of recombinant conopeptide production.

[0132] A variety of recombinant expression systems based on bacterial, yeast, insect or mammalian cells could be utilized for the production of the Conus PDI enzyme. The recombinant expression of conopeptide precursors, under ideal conditions, presents more stringent requirements. Ideally, a cellular expression system will be able to recognize the conopeptide precursor signal sequence and direct the nascent protein into the secretory pathway where proper processing, folding, secondary modifications and extra-cellular secretion of the bioactive peptide can occur. In such a system, the co-expression of the cloned Conus PDI will facilitate efficient folding of the disulfide-rich conopeptide and promote higher yields of correctly folded peptide. Alternatively, the recombinant PDI gene may be modified to provide a precursor signal sequence recognized by the host.

[0133] Both insect cell and mammalian cell expression systems have the potential to carry out the necessary processing steps for conopeptide expression and could serve as suitable hosts. Insect cells have an advantage in the fact that the cell lines are easier and less costly to maintain. In addition, many of the insect cell lines can be grown in serum-free media, which can greatly simplify the purification of secreted peptide products.

[0134] There are two general methods for recombinant protein production in insect cell lines, the baculovirus systems and the nonlytic, plasmid-based stable expression systems. While the baculovirus systems are capable of very high expression levels, the transient, lytic nature of the viral infection can interfere with the synthesis of secretory proteins, and the debris from virus-induced cell death can make purification of small secreted conopeptide products more difficult. The stable insect cell expression systems utilize a plasmid expression vector with a strong, insect cell-specific viral promoter to drive transcription of the cloned cDNA, along with a drug-selectable marker that permits selection of stable cell lines continuously expressing the recombinant protein of interest. These systems reportedly give more reliable expression of secreted protein products. The continuous nature of peptide synthesis, coupled with the secretion into serum-free media, will facilitate purification of the conopeptide products. For these reasons, the insect cell stable expression system is an advantageous method, although it is realized that other expression systems could be utilized for expression of the Conus PDI, either alone or co-expressed with conopeptide precursors.

[0135] The ˜1600 bp C. textile PDI cDNA is cloned into an insect cell expression plasmid under the control of the OpIE2 promoter, capable of providing constitutive expression in a variety of insect cell lines. The PDI cDNAs is engineered to contain a C-terminal fusion of the V5 epitope to allow antibody detection of the recombinant protein, and a 6× His tag to permit purification by metal affinity chromatography. A stable clonal cell line expressing the PDI enzyme is selected using an expression plasmid which also contains the blasticidine resistance gene under the control of the OpIE1 promoter. The PDI expression construct is introduced into an appropriate insect cell line (HighFive, Sf9, Sf21, for example) by liposome-mediated transfection. Blasticidine selection is used to isolate stable clonal lines that express the PDI enzyme. Following an incubation period of 2-4 days, cellular protein extract is analyzed by Western blotting, using anti-V5 antisera, to determine the relative expression levels of the recombinant Conus PDI protein being produced in the various cell lines. One or more cell lines that gives abundant expression of the PDI protein, as determined by Western blot, is selected. A high-expression cell line is used for purification of the Conus PDI protein by standard methods already established for purification of the enzyme from venom duct tissue. As an alternative, the recombinant His-tagged protein is purified by affinity chromatography on nickel resin columns. Once a stable cell-line is established and characterized, it can provide a continuous source from which to isolate the Conus PDI enzyme. Cell stocks can be frozen for future use, and cells can be adapted to nonadherent high-density growth in spinner flasks to enable large-scale production of the recombinant protein.

[0136] Generation of a stable cell line with high-level constitutive expression of the Conus PDI enzyme will facilitate efforts to co-express conopeptide precursors. A large number of conopeptide precursor genes have been isolated, the majority of which encode peptides with numerous disulfide bonds. Recombinant expression of these genes may allow facile production of the bioactive peptides. Co-expression of high levels of the PDI enzyme may promote efficient folding of the processed mature peptide.

EXAMPLE VI

Synthesis of Conopeptide in Host Cells Expressing Conus PDI

[0137] A Conopeptide precursor gene is cloned into a plasmid expression vector under the control of the OpIE2 promoter, to provide high-level constitutive expression in insect cell lines. The conopeptide genes encode a native signal sequence that should direct the nascent protein into the insect cell secretory pathway. Alternatively, the mature, bioactive conopeptide coding sequence is fused to the honeybee mellitin gene signal sequence, an insect-specific gene know to function as an efficient signal sequence in insect cell lines. The conopeptide gene is expressed as the native conopeptide sequence, or they can be engineered to contain a C-terminal fusion of an epitope tag or a 6×His affinity purification tag. While these C-terminal tags will permit efficient immuno-detection and purification of the recombinant conopeptide, the tag may interfere with the bioactivity of the peptide. In this case, a specific protease cleavage site is inserted between the conopeptide sequence and the tag sequence. In this embodiment, the conopeptide is purified using the tag and is then digested with a protease which cleaves at the inserted protease cleavage site. In specific instances, either alternative may be preferable. The expression construct may also contain the neomycin resistance gene, for dual selection of the conopeptide gene in the blasticidine-resistant PDI-expressing cell line. The conopeptide expression plasmid is introduced into the PDI-expressing cell line by liposome-mediated transfection, and the transfected cells is maintained in serum-free media. Following an incubation period of 2-4 days, culture media is harvested, concentrated, and analyzed for conopeptide production by gel electrophoresis, HPLC analysis or bioassay, depending on the particular conopeptide to be analyzed. The effectiveness of Conus PDI co-expression in improving bioactive conopeptide yield is determined by comparison to expression in native insect cell lines that lack the Conus PDI. For long-term conopeptide production, stable cell lines are selected by dual blasticidine-neomycin selection.

EXAMPLE VII

In Vivo Production of Recombinant PDI

[0138] The Conus PDI of SEQ ID NO:2 is modified by gene recombination technology so as not to code for the endoplasmic reticulum localization signal. The recombinant gene is linked to a highly active promoter, and introduced into host cells, which can multiply in a culture medium at nearly neutral pH, by transfection or transformation. For example, the recombinant gene is transformed into microorganism such as yeast, which is cultured in a culture medium free of a PDI activity inhibitor, such as a uracil-free minimal medium, with pH being kept nearly neutral using a buffer, for example, HEPES. A large amount of PDI having enzyme activity is expressed in the culture medium outside the cells.

[0139] An endoplasmic retention signal is modified by deleting, substituting, or adding one or more bases in a region encoding an endoplasmic reticulum localization signal of a gene encoding protein disulfide isomerase of Conus to modify the gene so as not to encode part, or all, of the endoplasmic reticulum localization signal. The modified PDI gene is positioned for expression in an expression vector and introduced into host cells. The host cells are cultured, thereby causing protein disulfide isomerase to be secreted in an active state outside the host cells.

[0140] In eukaryotic cells, secretion of proteins is performed usually along the following pathway: a secretory protein is translated from mRNA by the ribosome. During translation, the protein is transferred into the endoplasmic reticulum, and further transported to the Golgi apparatus, from which it is allocated through the secretory pathway to the vacuole, the cell membrane, and then the cell wall, or the outside of the cell. Signal sequences directing peptides to the secretory pathway are typically cleaved in the endoplasmic reticulum. Proteins, which should remain in the endoplasmic reticulum, also take the same route as the secretory protein, but once transported to the Golgi apparatus, they are transported back to the endoplasmic reticulum due to a special structure or sequence, in addition to a signal necessary for entry into the secretion pathway, such as a particular amino acid sequence comprising several residues (a consensus sequence), called a “motif” in which amino acid residues with similar properties may be substituted conservatively. The special structure or sequence necessary for persistent presence in the endoplasmic reticulum is designated an “endoplasmic reticulum localization signal.” For example, an endoplasmic reticulum localization signal may be a sequence, such as KDEL, located at the C-terminus of the protein.

[0141] Signal sequences maybe added to the protein disulfide isomerase, thereby allowing targeting of the PDI to the appropriate organelle of a host system. For example, the signal sequence of Conus PDI, may be replaced with a secretory signal sequence recognized by Sacchoromyces or any other organism, where desirable or appropriate, thereby directing the protein to the desired organelle. Further, where desirable and appropriate, both the endoplasmic reticulum localization signal and signal sequence may be modified to produce a protein disulfide isomerase which is secreted into the medium or other desired locations.

EXAMPLE VIII

[0142] PDI activity is measured by a sensitive fluorescent assay using the method of Heuck, A. P., and Wolosiuk, R. A. (1997) Anal. Biochem. 248:94-101. Briefly, 4-20 mg of PDI protein is incubated in 0.2 ml of a 0.1 mM sodium phosphate buffer (pH 7.4) containing 75 mM dithiothreitol, 3 mM EDTA, and 0.7 mM di-fluoresceinthiocarbamyl-insulin at 37° C. Fluorescence is monitored using a spectrophotometer.

[0143] Alternatively, protein disulfide isomerase activity is measured by refolding of “scrambled” RNase, which had exchanged cystines, as described elsewhere in Lambert, N. and Freedman, R. B. (1983) Biochem. J. 213:235-243. Bovine protein disulfide isomerase (Sigma) is used as a positive control.

EXAMPLE IX

[0144] To test for the presence and abundance of PDI in other Conus species, a simple PCR sceening was done, using the primer set of PDI-20F and PDI-21R. Species tested include: C. textile, C. stercusmuscarum, C. aurisiacus, C. consors, C. betulinus, and C. omaria. The PCR was done using, approximately Ing of venom duct cDNA, and the standard protocol of Taq polymerase. The reaction was run on a one percent agarose gel and then stained using ethidium bromide. As shown in FIG. 11 the expected ˜550 bp band is seen in each lane. Although it is clear that PDI is present in the venom ducts of all Conus species, the concentration is believed to vary as indicated by differences in band intensity shown in FIG. 11. The faint band for C. stercusmuscarum is believed to be due primarily to a problem with the template DNA used for the PCR.

EXAMPLE X

[0145] Full-length C. erminius, C. floridanus, C. geographus, C. gloriamaris, C. imperialis, C. magus, C. marmorceus, C. nigropunctatus, C. pennaceus, C. pennaceus, C. purpuracens, C. striatus, C. tulipa, or other Conus species PDI cDNAs are isolated by reverse transcription-PCR (RT-PCR) of venom duct RNA, using primers based on the highly conserved thioredoxin-like active site motif found in the PDI genes isolated from C. textile or other organisms. The PDI genes isolated from C. textile (SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5) contain two conserved repeats of this sequence motif, separated by ˜750-1000 bp of intervening sequence. A forward PCR primer is designed to match the N-terminal region sequence of the thioredoxin site as previously described, and a reverse primer is designed to the C-terminal thioredoxin site. These PCR primers will contain degenerate codon usage to account for DNA sequence variation in the corresponding Conus genes. Venom duct tissue is dissected from C. erminius, C. floridanus, C. geographus, C. gloriamaris, C. imperialis, C. magus, C. marmorceus, C. nigropunctatus, C. pennaceus, C. pennaceus, C. purpuracens, C. striatus or C. tulipa snails and used to prepare mRNA. The mRNA is reverse-transcribed into cDNA according to standard techniques. This venom duct cDNA is used for PCR amplification with the PDI forward and reverse primer pair previously described. PCR amplification is performed using standard methods known in the art and previously described to yield a PCR product. The PCR product is gel-purified, cloned into a plasmid vector, and several cloned isolates are sequenced. The open reading frame with significant homology to the PDI genes of SEQ ID NO:1 through SEQ ID NO:8 is identified. Using the above method, the open reading frame is predicted to extend between the two thioredoxin-like domains, and lack the 5′ and 3′ regions of the full-length cDNA. The DNA sequence of this initial PCR product is used to design nested PCR primers for 5′ and 3′ RACE procedures (rapid amplification of cDNA ends) to isolate the full-length cDNA. Venom duct cDNA from the appropriate species is synthesized with 5′ and 3′ RACE adapters and used for RACE amplifications. These 5′ and 3′ RACE products are gel-purified, cloned into a plasmid vector, and sequenced. The encoded PDI is determined based on the nucleic acid sequence.

[0146] Alternatively, The PDI sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, or a fragment thereof, is labeled, for example, with 32P. The cDNA library previously described is cloned into a vector and the vector library is transformed into a suitable host, for example, DH5α cells. The transformed cells are replica plated in triplicate and grown overnight. Three ofthe four plates are prepared for colony lift hybridization using standard techniques known in the art. The labeled nucleic acid is then hybridized to colony lift filters to identify clones containing the PDI genes. Positive clones are isolated, grown and the vector isolated. The isolated vector is sequenced.

[0147] While this invention has been described in certain embodiments, the invention can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the invention using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims. Thus, the described embodiments are illustrative and should not be construed as restrictive.

[0148] All references, includingpublications, manuscripts, patents, and patent applications, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Conus protein disulfide isomerase

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