COILED-COIL HETERODIMER METHODS AND COMPOSITIONS FOR THE DETECTION AND PURIFICATION OF EXPRESSED PROTEINS

Abstract: Methods and compositions employing complementary heterodimer-subunit peptides capable of forming a alpha -helical coiled-coil heterodimer for affinity purification and detection of fusion proteins.

Description: COILED-COIL HETERODIMER METHODS AND COMPOSITIONS FOR THE DETECTION AND PURIFICATION OF EXPRESSED PROTEINS This application claims priority to U S Patent Application Serial No 08/540.397 filed 6 October 1995. herein incorporated by reference Field of the Invention This invention relates to the use of a pair of peptides capable of forming α-hehcal coiled-coil heterodimers for the purification and detection of fusion proteins containing one of the peptides of the pair References Cited An, G , etal . Mol Cell Biol.2:1628-1632 (1982) Ausubel. F . et al . in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY. John Wiley and Sons, Inc , Media PA (1988). Brasier, A R , et al . BioTechmques 7:1116-1122 (1989) Clare, J J . et al , Bio/Technol 9455-460 (1991) Craxton, M , Methods- A Comparison to Methods Enzymol 320 (1991) Cregg. J.M , et al , Mol Cell Biol 5:3376-3385 (1985) Dykes, C W , et al . Eur J. Biochem 174411-416 (1988) Ellis. S.B , et al . 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Cold Spring Harbor Laboratory, Cold Spring Harbor, NY ( 1989) Saπn. et al . Anal Biochem J17 147-157 ( 1981) Skerra. A , et al , Biotechnology 9 273 ( 1991 ) Towbin. M . et al . Proc Natl Acad Sa USA 76 4350 ( 1979) Yamsch-Perron. C . et al . Gene 33 103-1 19 ( 1985) Background of the Invention One of the major commercial applications of biotechnology is recombinant expression of proteins Several recombinant proteins, including epoetm α, G-CSF. somatotropic, insulin, tPA, urokinase and prourokinase, various interferons, EPO, and specialty enzymes have well established commercial markets (Bioprocess Engineering) There are three general technical hurdles in bringing a protein to recombinant production The protein's coding sequence must be obtained and the gene cloned in a suitable expression vector, a host cell and culture conditions favorable for expressing and preferably secreting the protein in active form must be found, and the expressed protein must be isolated and, in some case, enzymatically processed, to produce a desired final product, e g , one suitable for pharmaceutical use For purposes of finding conditions that favor high levels of protein expression and secretion, it is desirable to be able to monitor expression, levels of expression, and localization of expressed proteins for a given expression system Ideally, such monitoring can be done without modifying the expressed protein and can be performed in an in situ format as well as various in vitto formats, such as dot blot or Western blot analysis For purposes of isolating expressed proteins, it is desirable to generate the protein in a form that lends itself to one-step affinity purification An approach that has been proposed previously for this purpose is to express the protein in tandem with a small peptide antigen segment One then purifies the expressed protein on the basis of its specific binding to a support matπx carrying an antibody specific against the antigen For large- scale production, this approach requires amounts of antibody commensurate with the amount of protein produced, which may add significantly to the cost of protein production The need for antigen competition to release the bound protein from the solid support may also add to the cost of the purification step Summary of the Invention The invention includes an expression vector composed of a replication segment which permits replication of the vector in a selected host cell, and an expression cassette The cassette contains, in a 5'-3' direction, a promoter functional in the host cell and a coding segment The coding segment, in turn, contains a heterologous DNA coding sue and a DNA region encoding a first heterodimer-subunit peptide capable of forming an α- helical coiled-coil heterodimer with a complementary second heterodimer-subunit peptide Further, the coding segment may contain, disposed between the coding site and the DNA region, a cleavage sequence, in frame with the DNA region and encoding an ammo acid sequence that provides a target for chemical or enzymatic cleavage The coding segment may be oriented with either its coding site or DNA region adjacent the promoter In a preferred embodiment, one of the (first and second heterodimer-subunit peptides) contains at least two heptad ammo acid repeat sequences having the form gabcdef, where positions a and d of each amino acid repeat sequence are selected from the group consisting of leucine, isoleucine and valine, and positions e and g of each amino acid repeat sequence are selected from the group consisting of aspartic acid and glutamic acid The other of the (first and second heterodimer-subunit peptides) contains at least two heptad amino acid repeat sequences having the form g'a'b'c'd'e' , where positions a' and d' of each amino acid repeat sequence are selected from the group consisting of leucine, isoleucine and valine, and positions e and g of each amino acid repeat sequence are selected from the group consisting of lysine, arginine and histidine In this preferred embodiment, each of the corresponding d/a' and a/d' pairs in complementary heptads consists of residues where one of the residues is a valine and the other is selected from the group consisting of leucine and isoleucine The cloning site preferably includes a multiple cloning site (MCS) at which a heterologous DNA coding region can be inserted Such a heterologous DNA coding region typically encodes a selected polypeptide of interest The DNA encoding the selected polypeptide is typically inserted at the cloning site such that it is expressed in frame with coding sequences in the DNA region encoding the first heterodimer-subunit peptide are the sequences represented by SEQ ID NO 2 and SEQ ID NO 4 In a related aspect, the invention includes a coding segment, for use in an expression vector suitable for expressing heterologous proteins in a host cell (such as the vector described above) The segment includes a heterologous DNA coding sue and a DNA region encoding a heterodimer-subunit peptide capable of forming an α-helical coiled-coil heterodimer with a complementary heterodimer-subunit peptide, as described above Further, the segment may contain, between the coding site and the DNA region, a cleavage sequence, in frame with the DNA region, encoding an amino acid sequence comprising a target for chemical or enzymatic cleavage The coding site may be situated either 5' or 3' of the DNA region The cloning site and coding sequences include the embodiments described above in relation to the expression vector Also included in the invention is a fusion protein encoded by the coding segment described above or produced by an expression vector containing such a coding segment, such as the expression vector described above The fusion protein is composed of a polypeptide having a functional activity (; e , a selected polypeptide of interest) and a heterodimer-subunit peptide (z e , a coil peptide, such as the E-coil, 0993, K-coil or 0994 peptide described herein) of the type described above, attached to the N- or C-terminus of the polypeptide Also included in the invention is a reagent for detecting the presence of the expressed fusion protein The reagent is composed of a second heterodimer-subunit peptide (second coil peptide) capable of forming an α-helical coiled-coil heterodimer with the heterodimer-subunit peptide (first coil peptide) in the fusion protein The reagent is useful for detecting expression of a fusion protein containing a heterodimer-subunit peptide, such as described above, by forming a heterodimer or heterodimer complex between the heterodimer-subunit peptide contained in the fusion protein and the detection reagent, and detecting the presence of heterodimer formation A kit for detecting the expression of a selected polypeptide includes an expression vector containing the above-described coding segment, for expressing a fusion protein containing the heterodimer-subunit peptide, and the above detection reagent In still another aspect, the invention includes an affinity matrix composition for the purification of a selected polypeptide The composition includes a solid support, and attached to said support, a heterodimer-subunit peptide (coil peptide, e g , SEQ ID NO 4 or SEQ ID NO 27) of the type described above The composition is useful in a method for purifying a selected expressed polypeptide, as described below The invention also includes a method of purifying a selected expressed polypeptide The method includes the steps of (I) expressing, in a suitable host-cell expression system (e g , an E coli or Yeast expression system), a fusion protein containing the selected polypeptide in tandem with a first heterodimer subunit (e g , SEQ ID NO 2 or SEQ ID NO 26), (n) obtaining, from the host cell expression system, a suspension containing said fusion protein, and (in) passing this suspension over an affinity matrix composition as described above The affinity matrix is then washed while the fusion protein remains bound to the affinity matπx via the α-hehcal coiled-coil heterodimer The washing includes, in either order, (a) a salt wash step comprising washing the affinity matrix with a solution having a pH of between about 5 5 and about 8 0 and containing between about 0 1 M and about 1 M salt (e g , sodium chloride, potassium chloride, sodium acetate, ammonium chloride, ammonium acetate, sodium perchlorate, potassium perchlorate, sodium phosphate and potassium phosphate), and (b) an organic wash step comprising washing the affinity matrix with a solution having a pH of between about 5.5 and 8 0 and containing between about 50% and about 100% of an organic solvent (e g , methanol, ethanol, isopropanol tetrahydrofuran, tπfluoroethanol, and acetonitrile) After washing, the selected polypeptide is released from the matrix either by elution or by enzymatic cleavage Preferably, the elution solution has a pH of about 3 0 or lower, or about 10 0 or higher Exemplary elution solutions contain between about 20% and about 80% of an organic solvent A kit for obtaining an expressed protein in purified form includes the above expression system, for expressing a fusion protein having a heterodimer-subunit peptide of the type described above, and an affinity matrix composition of the type described above, for affinity binding of the expressed fusion protein to the solid support in the composition These and other objects and features of the invention will be more fully appreciated when the following detailed descπption of the invention is read in conjunction with the accompanying drawings Brief Description of the Figures Figures IA, I B, IC, I D I E and IF show a scheme for affinity purification according to the method of the invention Figure 2 shows helical wheel representations of peptides 0993 (SEQ ID NO 26) and 0994 (SEQ ID NO 27) in an α-helical heterodimer configuration Figures 3A, 3B, 3C, 3D and 3E show a schematic representations of adjacent heptads of two heterodimer-subunit peptides in a parallel configuration comparing the stabilizing/destabilizing effects of charged residues at the e and g positions in homodimers vs heterodimers Figure 3A shows a homodimer stabilized by oppositely-charged residues at the e and g positions of a heptad Figure 3B shows a heterodimer destabilized by oppositely-charged residues at the e and g positions of a heptad Figure 3C shows a homodimer destabilized by positively-charged residues at the e and g positions of a heptad Figure 3D shows a heterodimer stabilized by like-charged residues at the e and g positions of a heptad Figure 3E shows a homodimer destabilized by negatively-charged residues at the e and g positions of a heptad Figures 4A, 4B and 4C show a schematic of some possible distributions of heptads, bearing either positive or negative charges at their e and g positions, within peptides designed to form coiled-coil heterodimers Figure 4A shows a schematic of a heterodimer comprised of heterodimer-subunit peptides having alternating positively- and negatively- charged successive heptads Figure 4B shows a schematic of a heterodimer comprised of heterodimer-subunit peptides, one of which has predominantly positively-charged heptads, and the other of which has predominantly negatively-charged heptads Figure 4C shows a schematic of a heterodimer comprised of heterodimer-subunit peptides. one of which has all positively-charged heptads, and the other of which has all negatively-charged heptads Figures 5A, 5B, 5C, 5D, 5E, 5F and 5G show a schematic of the synthesis of a DNA fragment encoding a heterodimer-subunit peptide Figure 5H shows a map of plasmid pRLD-E Figure 6A shows an expression vector expression cassette useful for expressing a fusion protein having a heterodimer-subunit peptide at the N-terminus of a selected protein encoded by a DNA fragment inserted at the multiple cloning site (MCS) Figure 6B shows an expression vector expression cassette useful for expressing a fusion protein having a heterodimer-subunit peptide at the C-terminus of a selected protein encoded by a DNA fragment inserted at the multiple cloning site (MCS) Figure 7A shows a reversed-phase (RP)-HPLC chromatogram of peptide 0993 (SEQ ID NO:26). Figure 7B shows an RP-HPLC chromatogram of the injection flow through fraction of a selective dimerization affinity column of the invention. Figure 8 shows an RP-HPLC chromatogram of the 60% acetonitrile wash fraction of the affinity column described in Figure 7B. Figure 9 shows an RP-HPLC chromatogram of a subsequent GndHCl elution fraction of the affinity column described in Figure 8. Figure 10 shows a chromatogram of a mixture of three test charged peptides (SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30) used to test specificity of the affinity matrix of the invention. Figure 1 1 shows a chromatogram of the 0.2 M KCl wash fraction of an affinity column onto which test peptides depicted in Figure 10 were loaded. Figure 12 shows a chromatogram of the 0.5 M KCl wash fraction of an affinity column to which test peptides were loaded. Figure 13 shows a chromatogram of the 1.0 M KCl wash fraction of the affinity column described in Figure 12. Figure 14 shows a chromatogram of a subsequent 1 .0 M KCl wash of the column described in Figure 13. Figure 15 shows a subsequent 5.0 M GndHCl/50mM K P0 4 of the column described in Figure 14. Figure 16A shows an SDS-PAGE gel of PAK( 128- 144)/E-coil fusion protein stained with comassie blue. Figure 16B shows western and ligand blots of PAK( 128- 144)/E-coil fusion protein probed with either coil K reporter or monoclonal antibody PK99H . Figure 17 shows a series of RP-HPLC chromatograms of load samples and fractions eluted from an affinity column of the invention, where the load fraction was a crude periplasmic extract containing recombinantly expressed E-coil peptide. Figure 18 shows a series of RP-HPLC chromatograms of purification of recombinantly expressed Pak-pili-E-coil peptide from crude on an affinity column made in accordance with the present invention. Figure 19 shows an ELISA assay employing a reporter molecule formed according to the invention. Detailed Description of the Invention I Definitions Unless otherwise indicated, the terms below have the following meaning The term ' peptide" designates a chain of amino acid based polyamides The chain can vary in length anywhere from 2 amino acids to about 100 ammo acids The term ' polypeptide" designates a chain of ammo acid based polyamides containing at least 2 ammo acids A promoter functional in a host means a DNA sequence in an expression vector effect to promote transcription of an adjacent 3' downstream coding sequence in the vector in a selected host A "coding segment" in an expression vector refers to a segment of DNA which encodes a polypeptide A "heterologous DNA coding site" refers to (I) a restπction sue or sues, e g , multiple cloning site, in an expression vector, into which a DNA that encodes a selected heterologous protein can be inserted, or (n) the encoding DNA itself A "heterodimer-subunit peptide" refers to one of two complementary peptide subunits which are capable of forming a coiled-coil heterodimer under selected conditions Further, the term "heterodimer polypeptide" or "heterodimer polypeptide complex" refers to two associated non-identical polypeptide chains Unless otherwise indicated, the sequence for peptides and polypeptides is given in the order from the am o terminus to the carboxyl terminus All amino acid residues identified herein are in the natural or L-configuration unless otherwise specified In keeping with standard peptide nomenclature, abbreviations for amino acid residues are standard 3- letter and/or 1 letter codes commonly used in the art The term "derivatized" , in the context of a "derivatized" polypeptide subunit, is understood to refer to a polypeptide subunit having one or more functional or reporter moieties covalently attached to one or more amino acid residues forming the subunit, where the moiety may be (l) coupled to one or more amino acid residues in the subunit either before or after polypeptide subunit synthesis, or (n) form an elongation of the peptide subunit, e g , at the subunit's N-terminus Further, the functional or reporter moiety may be attached to the polypeptide subunit directly, or through a linker or spacer, e g , a poly- glycine spacer The term "benign medium" as used herein, describes a physiologically-compatible aqueous solution typically having a pH of between about 6 and about 8 and a salt concentration of between about 50 mM and about 500 mM. Preferably, the salt concentration is between about 100 mM and about 200 mM. An exemplary benign medium, designated as buffer A. has the following composition: 50 mM potassium phosphate, 100 mM KCl, pH 7. Equally effective benign media may be made by substituting, for example, sodium phosphate for potassium phosphate and/or NaCl for KCl. II. General Overview of Heterodimer-Subunit Peptides Complementary heterodimer-subunit peptides are two non-identical peptide chains, typically about 21 to about 70 residues in length, having an amino acid sequence compatible with their formation into two-stranded α-helical heterodimeric coiled-coils. They are designated herein generally as HSP1 (heterodimer-subunit peptide 1 ), and HSP2 (heterodimer-subunit peptide 2). Because the peptides can form α-helical coils, they are also referred to herein as "coil peptides". In benign aqueous medium the isolated heterodimer-subunit peptides are typically random coils. When HSP1 and HSP2 are mixed together under conditions favoring the formation of α-helical coiled-coil heterodimers, they interact to form a two-stranded α- helical coiled-coil heterodimeric complex, designated as HSP1 - HSP2. Peptides in an α-helical coiled-coil conformation interact with one another in a characteristic manner that is determined by the primary sequence of each peptide: The tertiary structure of an α-helix is such that 7 amino acid residues in the pπmary sequence correspond to approximately 2 turns of the α-helix. Accordingly, a primary amino acid sequence giving rise to an α-helical conformation may be broken down into units of 7 residues each, termed heptads. The heterodimer-subunit peptides are comprised of a series of heptads in tandem. When the sequence of a heptad is repeated in a particular heterodimer-subunit peptide, the heptad may be referred to as a "heptad repeat", or simply "repeat" . As is detailed below, specific types of amino acid residues at defined positions in each heptad act to stabilize the two-stranded α-helical coiled-coil heterodimeric structure or complex. HSP1 and HSP2 may also contain residues that can be reacted (either intra- or inter- helically) to stabilize the α-helical or coiled-coil nature of the polypeptides. III. Features of Heterodimer-Subunit Peptides Complementary heterodimer-subunit peptides (HSP1 and HSP2) are typically of similar size, each generally ranging from about 21 to about 70 residues (3 to 10 heptads) in length The peptides may be synthesized by a variety of methods known to those skilled in the art, including chemical and recombinant methods. For example, an ABI Model 430A peptide synthesizer may be used with conventional t-Boc chemistry as described previously by Hodges, et al.. ( 1988), and in Example 1. Alternatively, the peptides may be expressed in an appropriate host-cell system using an expression vector encoding the peptides, as detailed, e.g. , in Examples 5 and 6. Subsequent to synthesis, the peptides may be purified by any of a number of methods known to those skilled in the art, for example using reversed-phase high performance liquid chromatography (RPC) and a "SYNCHROPAK" RP-P column, as detailed in Example 1. The composition and purity of the peptides can be verified by several methods, including amino acid composition mass analysis on a Beckman model 6300 amino acid analyzer and molecular weight analysis using time of flight mass spectroscopy on a "BIOION-20" Nordic, as detailed in Example 1. A. Coiled-Coil Heterodimer Formation The dimerization of HSP1 and HSP2 occurs due to the presence of a repeated heptad motif of conserved amino acid residues in each peptide's primary amino acid sequence. The individual positions in each heptad are designated by the letters a through g for HSP1. and a' through g for HSP2, as shown in Figure 2. The positions (e.g.. a', g') of HSP2 are sometimes referred to without the (') symbol in general discussions of heptad positions in heterodimer-subunit peptides, or coil peptides, below. Repeating heptad motifs having appropriate amino acid sequences direct the HSP1 and HSP2 polypeptides to assemble into a heterodimeric α-helical coiled-coil structure under permissible conditions, presented in part D, below. The individual α-helical peptides contact one another along their respective hydrophobic faces, defined as the a and d positions of each heptad. HSP1 and HSP2 may assemble into a heterodimer coiled-coil helix (coiled-coil heterodimer) in either parallel or antiparallel configurations. In a parallel configuration, the two heterodimer-subunit peptide helixes are aligned such that they have the same orientation (amino-terminal to carboxyl-terminal). In an antiparallel configuration, the helixes are arranged such that the amino-terminal end of one helix is aligned with the carboxyl-terminal end of the other helix, and vice versa Diagrams of the relative orientations ot the a-g positions of two interacting α- helices are shown in Figure 2 which shows an end-on schematic of the first two turns (one heptad) of two exemplary heterodimer-subunit peptides. 0993 (E, SEQ ID NO 26) and 0994 (K SEQ ID NO 27) arranged in a parallel configuration Heterodimer-subunit peptides designed in accord with the guidance presented herein typically show a slight preference for assembling in a parallel orientation vs an antiparallel orientation Generally, however, the orientation (parallel vs antiparallel) in which the two heterodimer-subunit peptides form an α-helical coiled coil is not particularly relevant to their ability to hold together moieties attached to the heterodimer-subunit peptides In Figure 2, amino acids are circled and indicated by the one-letter code, and consecutive amino acid positions are numbered and joined by lines with arrow heads indicating the N-terminal to C-terminal direction Interactions between the two helixes are indicated by arrows Wide arrows crossing between the helixes depict hydrophobic interactions between the a and d positions of adjacent helixes Ionic interactions between the e and g positions of adjacent helixes are indicated as curving arrows above and below the nexus of the helixes B Hydrophobic Interactions in Coiled-Coil Heterodimer Stability The hydrophobic interactions between the helixes are due to hydrophobic residues at the a and d positions of the heterodimer-subunit peptides Residues at these positions, effective to maintain the helixes in contact, include leucine, isoleucine. valine, phenylalanme. methionine, tryptophan, tyrosine, alanine and derivatives of any of the above Other residues, including alanine, cysteine, serine. threonine. asparagine and glutamine may also occupy a or d positions in some heptads, so long as others are occupied by hydrophobic residues Appropriate selection of the specific residues to occupy the a and d positions is an important aspect of the present invention If the hydrophobic interactions are too strong, a significant fraction of the helixes will form as homodimers at pH 7, even if like-charged residues are present at the e and g positions to discourage homodimer formation (see part C . below) If, on the other hand, residues at the a and d positions are selected such that the hydrophobic interactions are too weak (for example, Ala at both positions), the helixes may not form coiled-coil dimers at all Preferably, residue pairs at the a and d positions are selected to promote the formation 95% heterodimers at pH 7, more preferably > 99% heterodimers. The degree of heterodimer vs. homodimer formation may be measured as described. for instance, in Example 3. Results of experiments performed in support of the present invention indicate that if all the a and d positions in all heptads of both coil peptides are occupied by Ile. Leu. or any combination thereof, the hydrophobic interactions will be strong enough to stabilize homodimers as well as heterodimers. even if the e and g positions contained charged residues designed to stabilize heterodimers as detailed herein. Further, such homodimers will form among coil peptides containing as few as three heptads. According to an important aspect of the invention, the formation of such homodimers should be minimized or, for practical purposes, eliminated. The presence of a substantial fraction of homodimers would make practice of the invention difficult or impossible, since the coil peptides in the different component solutions (i.e. , the coil peptides in a suspension containing fusions of a selected protein and a coil peptide, or the coil peptides in a suspension to be used for derivatizing the solid matrix) would self-assemble before the component solutions could be used for their intended purpose. Several strategies may be employed to modulate or set the strength of hydrophobic interactions among residues at the a and d positions to a point where the interactions will be strong enough to maintain heterodimers during the protein purification procedure, yet not so strong that they will stabilize homodimers. An preferred strategy is to use coil peptides where all heptads have Leu at position d, and Val at position a, or coil peptides where all heptads have Ile at position d. and Val at position a. Another strategy is to use to use coil peptides where all heptads have Leu at position a. and Val at position d, or coil peptides where all heptads have Ile at position a, and Val at position d. Variations of these strategies were employed in the design of exemplary heterodimer-subunit peptides E-coil (SEQ ID NO:2) and K-coil (SEQ ID NO:4), as well as peptides 0993 (SEQ ID NO:26) and 0994 (SEQ ID NO:27). Yet another strategy is to occupy all but one of the a or d positions in each coil peptide by Ile and Leu, and use a destabilizing residue, such as Ala or Asn, in the remaining a or d position in each coil peptide. This remaining position is preferably closer to the center of each coil peptide than to either end. C. Ionic Interactions in Coiled-coil Heterodimer Stability Dimeric coiled-coil conformations of α-helixes can be stabilized by ionic interactions between residues at the e and g positions of adjacent helixes, as is illustrated in Figure 3. Other factors being equal, if each helix of a dimer has a positively-charged residue at one position, for example, e, and a negatively-charged residue at the other position, for example, g. homodimer formation is favored (Fig. 3A; compare with heterodimer in Fig. 3B). However, if each helix has like-charged residues at both positions, then two oppositely-charged helixes will tend to associate into heterodimers (Fig. 3D), as opposed to forming homodimers (Fig. 3C. 3E). The conformation of polypeptides, such as HSP1 and HSP2, in solution can be determined from CD spectra of the solution. These data provide information as to the conformation of the individual peptides themselves (random coil vs. α-helical). as well information as to the relative amounts of heterodimer vs. homodimer complexes of, for example, HSP1 and HSP2. Example 2 details one method of measuring CD spectra. Example 3 details how a CD spectra measurements can be used to assess the conformation of peptides in solution. In the diagram shown in Figure 2, ionic interactions between the two helixes arise from negatively-charged (Glu) residues at the e and g positions on HSP1 (0993; SEQ ID NO:26), and positively-charged (Lys) residues at the e and g positions on HSP2 (0994; SEQ ID NO:27). Negatively-charged residues can be aspartic acid, glutamic acid or derivatives thereof. Positively-charged residues can be lysine, arginine. histidine. or derivatives thereof. D. Conditions Favorable for Coiled-coil Formation Heterodimer-subunit peptides comprised of repeating heptads and designed according to the guidance presented in parts A through C. above, will readily form coiled- coil heterodimers in a benign medium, defined above in part 1. The degree of α-helical coiled-coil heterodimer formation can be determined from CD spectra, as described, for instance, in Example 3. Coiled-coil heterodimers may form under conditions outside the pH and salt range given for a benign medium, but some of the molecular interactions and relative stability of heterodimers vs. homodimers may differ from characteristics detailed above. For example, ionic interactions between the e and g positions that tend to stabilize heterodimers may break down at low or high pH values due to the protonation of, for example, Glu side chains at acidic pH, or the deprotonation of, for example, Lys side chains at basic pH. Aforementioned effects of low and high pH values on coiled-coil heterodimer formation may be overcome, however, by increasing salt concentration. Increasing the salt concentration can neutralize the stabilizing ionic attractions or suppress the destabilizing ionic repulsions Certain salts have greater efficacy at neutralizing the ionic interactions For example, a I M or greater concentration of C10 4 anions may be sufficient to induce maximal α-helical structure (as determined by CD measurements performed as detailed in Example 2), whereas a 3M or greater concentration of Cl " ions may be required for the same effect The effects of high salt on coiled-coil formation at low and high pH also show that mterhelical ionic attractions are not essential for helix formation, but rather, are a factor in whether a coiled-coil tends to form as a heterodimer vs a homodimer E Heptad Variation in Heterodimer-Subunit peptides Parts A, B and C, above, present guidelines as to which amino acid residues may be included, and which amino acid residues are preferable, at specific positions in heptads of heterodimer-subunit peptides that will typically result in those peptides forming α-helical coiled-coil structures in a benign medium This part describes some examples of how heptads with sequences which are in compliance with the guidelines presented in parts A through C, above, can be arranged within the heterodimer-subunit peptides Heterodimer-subunit peptides of the present invention may each contain from three to a plurality of heptads The sequences of each of those heptads may all be the same, or they may differ Furthermore, the sequences of the internal repeats may differ from one another depending on, for example, whether or not the repeats incorporate amino acid coupling residues, such as cysteine Such a coupling residue may be used, for example, to anchor a heterodimer-subunit peptides to a resin support matπx or to another polypeptide Because the salient interactions between two heterodimer-subunit peptides in an α- helical coiled-coil heterodimer pair are between adjacent, "complementary" heptads in each peptide, the primary sequence of heptads within a heterodimer-subunit peptide can vary, so long as the residues within each heptad interact favorably with residues in the complementary heptad of the second heterodimer-subunit peptide It follows, then, that adjacent heptads may vary in sequence such that, for example, the net charge on the heterodimer-subunit peptides can be altered without affecting the ability of the polypeptides to form α-helical heterodimer coiled-coils This relationship is illustrated in Figure 4 The figure shows three examples of CP dimer pairs Each heterodimer-subunit peptide has 5 heptads The + or - symbols in each heptad each represent two charges (one at the e position and one at the g position) Note that adjacent complementary heptads have opposite charges For the purpose of this example, it is assumed that positions other than e and g in each heptad sum to a net charge of zero It can be appreciated that HSPl and HSP2 forming the dimer in Figure 4A have net charges of +2 and -2, respectively due to an excess of one positively-charged heptad. and one negatively-charged heptad. respectively Similarly HSPl and HSP2 in Figure 4B have net charges of +6 and -6, respectively, and HSPl and HSP2 in Figure 4C have net charges of + 10 and -10 respectively Other variations on this theme are. of course, possible without departing from the spirit of the invention G Moiety Coupling to Heterodimer-Subunit Peptides Coupling residues, such as amino-acid coupling residues, may be incoφorated into one or both coil peptides to facilitate subsequent uses of the peptides The coupling residues may be incoφorated at in the central region of the peptide and/or at one or both ends In cases where the coupling residues are in the central region, the residues are typically incoφorated at positions b, c and/or f, preferably at position f, of one or more heptads These positions lie along the outward face of a coiled-coil heterodimer Coupling residues at one or both ends are typically coupled to the terminal residue Preferred couphng groups are the thiol groups of cysteine residues, which are easily modified by standard methods Example 4 details how the cysteine thiol groups present in coil peptides can be used to attach other peptides at those positions Other useful coupling groups include the thioester of methionine, the imidazolyl group of histidine, the guamdiny l group of arginine. the phenolic group of tyrosine and the mdolyl group of tryptophan These coupling groups can be derivatized in a manner similar to that detailed in Example 4, using reaction conditions known to those skilled in the art H Exemplary Coil Peptides The E-coil (SEQ ID NO 2) and K-coil (SEQ ID NO 4) heterodimer-subunit peptides, as well as the 0993 (SEQ ID NO 26) and 0994 (SEQ ID NO 27), are exemplary HSPl and HSP2 heterodimer-subunit peptides Both peptides contain Val residues at their a positions, and Leu residues at their d positions, ensuring hydrophobic interactions effective to stabilize coiled-coil heterodimers, but not strong enough to overcome the electrostatic repulsion between homodimers The e and g positions of 0993 heptads contain Glu residues, whereas the e and g positions of 0994 heptads contain Lys residues The opposite charges at corresponding positions within complementary heptads of 0993 and 0994 stabilize α-hehcal coiled-coil heterodimers, as was described above and illustrated in Figs 3A-E and 4 ln an analogous manner the charged groups at the e and g positions discourage the formation of, and destabilize, homodimers According to an aspect of the present invention, this destabihzation is strong enough to overcome the hydrophobic interactions present between appropriately-chosen residues at the a and d positions, which favor the formation of both heterodimers and homodimers As stated above, the salient interactions between two heterodimer-subunit peptides in an α-helical coiled-coil heterodimer pair are between adjacent, complementary heptads in each peptide In a preferred embodiment of the invention, each of the corresponding d/a' and a/d' pairs in such complementary heptads consists of residues where one of the residues is a valine and the other is selected from the group consisting of leucine and isoleucine This relationship can be appreciated in Fig 2, where the d/a' pair consists of Leu (at the d position) and Val (at the a' position), and the a/d' pair consists of Val (at the a position) and Leu (at the d' position) IV Protein Expression Vectors Employing Heterodimer-Subunit Peptides for Fusion Protein Purification The invention includes an expression vector useful for expressing a fusion protein of a selected polypeptide expressed in tandem with a coil peptide (I e, a heterodimer-subunit peptide) The coil peptide portion of the expressed fusion may then be used to purify the protein using an affinity matrix (as described below), or to detect the protein, either in situ (e g . fluorescence detection in a cell) or in a protein detection system, such as enzyme- linked immunosorbent assay (ELISA). and/or a dot. slot or Western blot The fusion protein is detected using a detection reagent which includes, e g , a reporter-labeled coil peptide complementary to the coil peptide in the fusion Methods for cloning selected sequences into expression plasmids or vectors are known to those skilled in the art (e g , Maniatis, et al , 1982, Ausubel, et al , 1988) Such plasmids or vectors may then be transformed into suitable host cells, such as bacteria or yeast, and the cells induced to produce recombinant polypeptides Depending on the expression system, the medium in which the cells were induced, or the cells themselves, are used to make a suspension containing the recombinant polypeptide, which may then be purified using the methods of the invention By way of illustration, Example 5 details the recombinant construction of the E-coil gene using two sets of complementary overlapping oligonucleotides The oligonucleotides in each set were annealed, digested with the appropriate restπction enzymes to create sticky ends at one end of each double-stranded (ds) fragment (Figs. 5C and 5F), and the two ds fragments were ligated with T4 DNA Ligase to generate a single ds fragment (Fig. 5G) of 177 base pairs. The 177 bp fragment was purified and digested with EcoRI and BamH] to produce a 166bp fragment suitable for cloning into a plasmid vector. The construction of an exemplary plasmid expression vector, pRLD. is described in Example 6. pRLD was constructed by changing the polylinker site of expression vector pASK40 (Skerra, et al.. 1991 ) to correspond to the polylinker of pHIL-Sl and PIC9 (Invitrogen, San Diego, CA). This modification was designed to shift the reading frame and make pASK40's cloning site more consistent with that of Pichia pastoris vectors. A sequence encoding the E-coil gene was then cloned into pRLD to produce the E. coli E-coil vector pRLD-E (Figure 5H). The expression vector may contain a replication segment which permits autonomous replication of the vector in a selected host cell, and an expression cassette. The cassette contains, in a 5'-3' direction, a promoter functional in the host cell and a coding segment, which is described in detail, below. The vector may also contain additional elements, such as sequences encoding a selectable marker that assure maintenance of the vector in the cell, an appropriately positioned ribosome-binding site downstream of the promoter, and transcription termination (TT) sequences at the 3' end of the cassette. The elements preferably contained in expression vectors depend on the particular expression system with which the vectors are used. For example, elements typically present in E. coli expression vectors include (i) sequences encoding a selectable marker that assure maintenance of the vector in the cell (e.g. , an ampicillin resistance gene), (ii) a controllable transcriptional promoter which, upon induction, can produce large amounts of mRNA from a selected gene cloned into the vector, (iii) translational control sequences, such as an appropriately positioned ribosome-binding site and initiator ATG, and (iv) a polylinker or multiple cloning site (MCS) to simplify the insertion of the selected gene in the correct orientation within the vector. Examples of inducible promoters suitable for use with E. coli expression vectors include lac, trp, and tac, which, upon induction, can produce large amounts of mRNA from genes operatively linked to the promoters. Other promoters suitable for use with E. coli expression vectors include the highly-efficient phage T7 gene 10 promoter, which uses T7 RNA polymerase, and the powerful bacteriophage pL promoter (Ausubel, et ai , 1988). Recombinant polypeptides or fusion proteins are typically expressed by transforming a suitable E. coli expression strain (e.g. , JM83) with expression plasmids encoding recombinant polypeptides or fusion proteins Examples of suitable transformation methods include heat-shock treatment (Yanisch-Perron. et al , 1985) and electroporation The transformed cells are then typically plated on selective agar plates (<? g . LB plates supplemented with carbenicillin ( 100 μg/ml)), and cells containing the appropπate recombinant DNA are verified by restriction digest analysis and subsequent DNA sequencing of minipreped plasmid DNA Selected E coli clonal cells harboring the appropriate DNA are typically grown (e g , at 25 °C with shaking in LB medium containing 100 μg/μl of carbenicillin) to a suitable density (e g , an A 550 of 0 5) When using plasmids requiring induction, e g , with IPTG, the cells are induced by addition of the appropriate inducing agent A suspension containing the expressed protein may be obtained using methods known in the art, e g , a modified osmotic shock treatment (Ausubel, et al , 1988) Although proteins expressed in large amounts in E coli sometimes precipitate into insoluble aggregates (inclusion bodies), such proteins may be recovered using, for example, solubilization in denaturing agents known to those skilled in the art (Ausubel. et al , 1988) Other types of host cell expression systems may be employed in the practice of the invention For example, yeast, such as Pichia pastoris, offers a number of advantages, including very high expression levels, ease of use and some postranslational modification Yeast expression vectors may be used to transform yeast spheroplasts, and the transformed cells used to produce recombinant polypeptide Exemplary inducible promoters suitable for use with yeast expression vectors are the AOX promoter, described above, and the GALL GAL4, GAL7 and the GAL10 promoters Example 7 describes the use of a yeast expression vector in the practice of the invention A DNA fragment containing the E-coil gene was PCR-amplified and cloned into plasmid pIC9 (Invitrogen, San Diego, CA), generating Pichia E-coil vector p9CE Verified yeast plasmids were transformed into Pichia pastoris, and the yeast cells were selected, induced and processed as described in Example 7 to generate culture supernatant containing the recombinant protein Expression cassettes or coding segments of the present invention may also be used to express fusion proteins usmg the baculovirus system, where genes for proteins to be expressed are inserted into an insect virus in lieu of a highly expressed dispensable gene The foreign protein is produced by growing the recombinant virus in cultured insect cells (Ausubel, et al , 1988). Such vectors typically contain recombination sequences to allow the vector to integrate into the host cell's genome via homologous recombination The specific promoters used in these vectors generally depend on the identity of the excised dispensable gene. Exemplary promoters include the polyhedrin promoter (normally used to drive expression of the AcMNPV polyhedrin gene) and the plO promoter (Ausubel. et ai , 1988). Mammalian cells may also be used as host cells for vectors employing expression cassettes or coding segments of the present invention. Two exemplary mammalian systems employ the COS and CHO cell lines, respectively, ln the COS system, vectors containing the gene to be expressed are transiently transfected into COS cells, which constitutively produce SV40 large T antigen. Vectors used with COS cells typically contain an SV40 replication origin, such that when the vectors are transfected into COS cells, they replicate and thereby amplify the amount of protein expressed. CHO cells may be stably transfected with vectors employing expression cassettes or coding segments of the present invention and carrying either the dihydrofolate reductase or the glutamine synthetase gene (whose products confer drug resistance). Cell lines that carry increased numbers of the constructs are obtained by selecting those that grow in increasing drug concentrations (e.g. , methotrexate). Once selected, these lines are permanent reagents, which can be stored frozen and used to produce the protein whenever desired. Coding Segment The coding segment contains a heterologous DNA coding site, a DNA region encoding a first heterodimer-subunit peptide capable of forming an α-helical coiled-coil heterodimer with a complementary second heterodimer-subunit peptide. and disposed between the coding site and the DNA region, a cleavage sequence. The cleavage sequence is in frame with the DNA region, and encodes an amino acid sequence that provides a target for chemical or enzymatic cleavage. The cleavage sequence is preferably included in coding segments of the present invention to facilitate the removal of the coil peptide from its fusion partner (the selected polypeptide of interest). However, it will be understood that in applications where it is not desired to remove the coil peptide from the fusion protein, the cleavage sequence is not necessary. In this respect, the invention contemplates a coding segment containing only a heterologous DNA coding site and a DNA region encoding a first heterodimer-subunit peptide capable of forming an α-helical coiled-coil heterodimer with a complementary second heterodimer-subunit peptide. In a general embodiment, the coding site is a multiple cloning site (MCS), also referred to as a polylinker or polylinker site. The MCS typically contains a number of restriction enzyme sites useful for cloning selected polynucleotides to be expressed into the coding segment or vector containing the coding segment Examples of suitable poly nkers can be found in virtually all commercially-available cloning vectors (e g . vectors from Stratagene. La Jolla, CA or Invitrogen. San Diego, CA) In other embodiments, the coding site contains a gene encoding a protein of interest The generation and incoφoration of such genes into expression vectors of the invention is described in Examples 9 and 10. respectively In Example 9, a gene encoding PAK pi antigen is generated by annealing synthetically-produced complementary oligonucleotides and cloning the ds fragment into appropriate restriction sues in the pRLDE vector In Example 10, a cDNA encoding GFP is cloned into pRLDE Exemplary proteins which may be purified using the methods of the present invention include epoetm α, G-CSF, somatotropic, insulin, tPA, urokinase and prourokinase, vaπous interferons, EPO, and various specialty enzymes The coding segment may contain the coding site oriented either 5' or 3' of DNA region These two possibilities are illustrated Figs 6A and 6B, which show schematics of expression cassettes of the present invention The cassettes contain a promoter functional in the selected host cell system, and, disposed at the 3' ends of the promoter regions, coding segments as described above In Figs 6A and 6B, the heterologous DNA coding sites are represented as "cDNA" and the DNA regions encoding a first heterodimer-subunit peptide are represented as "Coil DNA" Figure 6A shows the coding sue oriented at the 3' end of the DNA region, whereas Figure 6B shows the coding sue oriented at the 5' end of the DNA region Cleavage sites that constitute targets for rare-cutting proteases are known in the art (Ausubel. et al . 1988) Such proteases mclude factor Xa (Nagai and Thogersen, 1984, 1987), thrombin (Smith and Johnson, 1988, Gearing, et al , 1989), enterokinase (Dykes, et al , 1988, LaVallie. et al , 1993), renin (Haffey, et al , 1987), and collagenase (Germino and Bastia, 1984) Factor Xa and enterokinase are particularly useful because they cleave on the carboxy-terminal side of their respective recognition sequences, allowing the release of fusion partners containing their authentic ammo-termini The recognition sites for enterokinase, factor Xa and thrombin are provided herein as SEQ ID NO 21 , SEQ ID NO 23 and SEQ ID NO.25, respectively The primary reason for providing coding segments having the two different arrangements of elements described above is that fusions of a peptide (such as a coil peptide) with a selected polypeptide of interest sometimes alter a desired activity or characteristic of the polypeptide of interest By generating two different fusion, one of which has the coil peptide at the N-termmus, and the other of which has the coil peptide at the C-terminus of the selected polypeptide of interest the probability of obtaining a fusion that does not significantly alter a desired property or characteristic of the selected polypeptide of interest is increased Exemplary coding sequences for the DNA region encodmg the first heterodimer- subunit peptide are the sequences represented by SEQ ID NO 2, SEQ ID NO 4. SEQ ID NO 26 and SEQ ID NO 27 It will be appreciated that in the case illustrated in Fig 6A where the DNA region is upstream of the coding sue, the DNA region typically includes an initiation ATG codon as s first codon In cases such as illustrated in Fig 6B, where the DNA region is downstream of the coding sue, the initiation ATG codon is typically provided by the DNA encoding the polypeptide of interest inserted at the coding site Coding segments of the present invention are preferably provided in three configurations for each of the two arrangements of elements described above The three forms each differ from one another by having different-length nucleotide spacers between the coding site and the cleavage site The spacers are included to provide coding segments having the coding sue in all three reading frames relative to the reading frame of the DNA region and the cleavage site In this way, an appropriate coding segment can be selected by those of skill in the art to express any DNA fragment having ends compatible with restπction sues present at the MCS in frame with the DNA region and the cleavage sue V Affinity Matrix Composition Employing Heterodimer-Subunit Peptides for Fusion Protein Purification The invention also includes an affinity matrix composition useful for purifying polypeptides, such as fusion proteins containing selected polypeptides expressed in tandem with a first heterodimer-subunit peptide The matrix includes a solid support having attached thereto a heterodimer-subunit peptide that is capable of forming an α-helical coiled- coil with another, complementary heterodimer-subunit peptide expressed as part of a fusion protein The affinity feature of the matrix is based on selective dimerization of the complementary heterodimer-subunit peptides The selective dimerization process for purification is shown schematically in Figures 1A-1F Figure IA shows a selective dimerization matπx 30 consisting of solid phase matrix 32 and heterodimer-subunit peptide 34 Optionally matrix 32 is conjugated to peptide 34 through a spacing group 36 The presence of such a group may be particularly advantageous when the protein to be purified on the column is a large or sterically bulky protein Solid phase matrix 32 may be formed by any of a number of suitable materials known in the art Preferably, the solid phase material will consist of spherical particles such as aminopropyl-deπvatized glass particles, capable of forming a packed matrix in a chromatography column, however, the solid phase may be formed by a continuous surface or by non-spherical particles or beads The method used for conjugation of the peptide to the solid phase will be readily determined by the skilled practitioner, based on the type of matπx. the type of spacing group, if present, and the desired peptide linkage The fusion protein is puπfied by first obtaining, from the host cell expression system, a suspension containing the fusion protein using methods known in the art (e g , Ausubel, et al ) This suspension is typically an impure solution containing impurities, such as other proteins, in addition to the desired fusion protein With reference to Figure IB, for purification of such a fusion protein from an impure solution containing other proteins, it can be seen that as the suspension is passed over the affinity matrix composition, the affinity matπx 30 binds to fusion protein 38 containing selected polypeptide 40. and heterodimer-subunit peptide 42. complementary to the immobilized heterodimer-subunit peptide 34 Also shown are non-binding components of the solution such as impurities 44 that do not specifically interact with the heterodimer- subunit peptide on the column These impurities are removed from the column milieu by extensive washing of the column Exemplary wash conditions include a salt wash step with a solution having a pH of between about 5 5 and about 8 0 (e g . 6 0) and containing between about 0 1 M and about 1 M (e g . 0 5 M) salt (e g . NaCI). and an organic wash step with a solution having a pH of between about 5 5 and 8 0 (e g , 6 0) and containing between about 50% and about 100% (e g , 75 %) of a suitable organic solvent (e g , acetonitrile) Suitable salts include sodium chloride (NaCl), potassium chloride (KCl), sodium acetate (NaOAc). ammonium chloride (NH 4 C1), ammonium acetate (NH 4 OAc), sodium perchlorate (NaOC10 4 ), potassium perchlorate (KOC10 4 ), sodium phosphate (Na : HP0 4 ), and potassium phosphate (K 2 HP0 4 ) Suitable organic solvents include methanol (MeOH), ethanol (EtOH), isopropanol, tetrahydrofuran (THF), tπfluoroethanol (TFE), and acetonitrile ALthough the order in which the salt and organic washes are performed is not critical, if the organic wash is done first, the column should be rinsed well with an aqueous solution (e g , water) before the start of the salt wash, to remove as much of the residual organic solvent as possible For this reason, it is generally preferable to do the salt wash first, followed by the organic wash, followed by a final aqueous rinse before the desired protein is eluted from the column. Figure IC shows a schematic of the column matrix after a wash step. Impurities shown in Figure IB are now absent from the column milieu. Fusion proteins 38 are now bound to the column through an interaction between immobilized heterodimer-subunit peptide 34 and complementary subunit 42 that forms a part of fusion protein 38. Figures ID and 1E-F show alternative means of eluting selected protein 40 from the column (i.e. , means for releasing the selected polypeptide from the matrix). Figure IE depicts a scheme in which the fusion polypeptide 38 is eluted from the column by washing with a buffer, such as a highly ionic buffer, that disrupts the interactions between the subunits of the coiled-coil heterodimer. As illustrated, fusion protein 38 has been removed from the column by contact with such a buffer. As is detailed below, a suitable ionic buffer is 0.5 M guanidium chloride. Alternatively, the fusion protein may be removed by eluting with a solution having a pH of less than about 3.0 and containing between about 20% and about 80% (e.g. , 50%) of a suitable organic solvent (e.g.. acetonitrile). The low pH of such an elution solution results in protonation of the negatively-charged residues (e.g.. Glu). disrupting the ionic interactions that stabilize the heterodimer. In another embodiment, the fusion protein is eluted with a solution having a pH of more than about 10.0 and containing between about 20% and about 80% (e.g. , 50%) of a suitable organic solvent (e.g. , acetonitrile). The high pH of such an elution solution results in neutralization of the positively-charged residues (e.g., Lys), disrupting the ionic interactions that stabilize the heterodimer. To complete the purification process, selected polypeptide 40 may be cleaved (Fig IF) from complementary heterodimer-subunit peptide 42 by a peptide cleavage reaction, such as by enzymatic cleavage. As an alternative, selected polypeptide 40 is directly removed from the column by cleavage in situ, as by enzymatic cleavage. As illustrated in Figure ID, following cleavage, the polypeptide divides into complementary subunit portion 42, which remains bound as a coiled-coil heterodimer to column matrix heterodimer-subunit peptide 34, and selected protein or polypeptide 40. From the foregoing it will be seen that the selective dimerization method of the invention is a useful and efficient means for purifying proteins, particularly fusion proteins, containing a heterodimer-subunit peptide capable of forming an α-helical coiled-coil in accordance with the present invention. Examples 8 and 12 describe the preparation and use of an affinity matrix designed for purification of polypeptides containing a coil peptide including exemplary coupling protocols to attach one of complementary heterodimer-subunit peptides to the matrix, preparation of an affinity column using the derivatized matπx, analysis of complementary heterodimer-subunit peptides and/or suspensions containing various contaminants along with such peptides as fusions with selected proteins run through such a column, and removal of the selected peptides and/or fusions from the column Example 1 1 describes results of Western and ligand blot analyses of proteins purified using the above-described method Figure 16A shows an image of a gel stained with Coomassie blue showing the relative purity of PAK (128- 144)/E-coιl puπfied using a K-coil affinity column The gel was blotted and subjected to Western blot and ligand blot analyses as described in the Example The results, shown in Figure 16B, show that methods of the invention can be successfully used to effectively purify recombinant fusion proteins or polypeptides VI Detection Reagents Employing Heterodimer-Subunit Peptides The invention includes synthesis and use of a reagent for detecting expressed proteins, in particular fusion proteins having a heterodimer-subunit peptide capable of forming an α-helical subunit in conformance with the invention The reagent can be used in a number of research and clinical applications, including, but not limited to detection of expression of fusion proteins, ELISA applications, radioimmunoassays and the like The detection reagent may also form part of a kit for detecting the expression of a selected polypeptide, where the kit also includes an expression vector that includes a DNA region encoding a heterodimer-subunit peptide capable of forming an α-helical coiled-coil heterodimer in accordance with the present invention In one form, the detection reagent includes (i) a heterodimer-subunit peptide designed and formed in accordance with the methods described herein, and (n) a reporter molecule The reporter molecule may be coupled to the heterodimer subunit peptide (e g , coupled to an amino acid coupling residue, such as a cysteine residue, at an outwardly- facing heptad position (e g , the f position) of the heterodimer-subunit peptide, or it may be synthesized, for example, in tandem with the coil peptide (e g . as described in Example 10) Alternatively reporter molecule may be incoφorated into the heterodimer subunit, for example as a radioactive amino acid Radioactive amino acids are commercially available from a number of sources and can be introduced into the heterodimer-subunit peptide during chemical or biological synthesis, according to methods well known in the art The reporter molecule can be selected from a number of reporter molecules well known in the art. including but not limited to radioactive groups, fluorescent tags enzymes and the like Use of enzyme markers and methods of incoφorating them into biological detection systems are known in the art (see, for example, Ausubel, et al ( 1988). for general methods, for specific exemplary reporter enzymes, see, for example the following luciferase (Brasier, et al , 1989, Gould and Subramani, 1988), chloramphenicol acetyltransferase (CAT, Gorman, et al , 1982), /3-galactosιdase (An, et al , 1982)) Preferably, the reporter molecule is attached to a coupling position in the heterodimer- subunit peptide that does not interfere with heterodimer formation One preferred position is position f of the subunit Example 13 provides details for forming a heterodimer-subunit peptide reporter molecule in accordance with the invention Figure 19 depicts a reporter molecule in a solid phase (ELISA) assay in a specific embodiment used to detect the presence of expressed proteins in accordance with the present invention In one representative embodiment, the reporter molecule is employed in an ELISA detection system used to quantitatively detect the presence of a heterodimer-subunit fusion protein, such as the PAK pili antigen/E-coil fusion protein described in Example 9 Figure 19 shows a schematic diagram of an ELISA detection system to detect the a fusion protein formed from a 17 amino acid Pseudomonas aeruginosa strain PAK pilin- deπved peptide Referring to the figure, a solid phase such as plate 50 is coated with a capture molecule such as PAK pilin peptide-specific antibody PK99H 52, according to standard methods known in the art A sample containing fusion protein 54 is contacted with the plate under conditions that promote binding of the protein to antibody 52 As illustrated fusion protein 54 is composed of a heterodimer-subunit peptide 56 and PAK pihn peptide 58 Following washing of the plate to remove unbound proteins, detection reagent 60, composed of a complementary heterodimer-subunit peptide 62 attached to reporter molecule 64, is added to the plate and allowed to react, under conditions that promote formation of a coiled-coil heterodimer complex between the complementary heterodimer-subunit peptides 56 and 62 Following further washing of the plate, detection of reporter molecule is used to quantitate the amount of fusion protein 54 bound to the plate Other embodiments of the invention using the reporter molecule will be apparent from the description provided above For example, the reagent can be used to detect the presence of a complementary heterodimer-subunit peptide-containing fusion protein in a gel or on a blot likewise the reagent can be used to detect expression of the fusion protein by plaques or in cell expression systems, according to methods known in the art The following examples illustrate, but in no way are intended to limit the present invention Materials and Methods A Abbreviations Abbreviations used in the Examples are t-BOC, tertiary butoxycarbonyl, DCM, dichloromethane, DIEA, diisopropylethylamine, DCC, dicyclohexylcarbodimide, DMF. N,N-dιmethylfoπnamιde. Gnd, guanidinium, HF, hydrogen fluoride. DCU, dicyclohexylurea. BSA. bovine serum albumin B Buffers Phosphate-buffered saline (PBS) lOx stock solution, 1 liter 80 g NaCI 2 g KCl 11 5 g Na,HP04-7H-,0 2 g KH,P0 Working solution, pH 7 3 137 mM NaCl 2 7 mM KCl 4 3 mM Na,HP0 4 -7H : 0 1 4 mM KH P0 4 C Strains and Plasmids E coli expression strain JM83 (Yanisch-Perron, et al , 1985) obtained from (New England Biolabs, Beverly, MA) was used as the host cell in experiments exemplifying a prokaryotic expression system Cells were cultured in Luna broth (LB) media (Miller, 1972) supplemented with 100 μg/ml of carbenicillin Plasmids for the eukaryotic system were propagated and cloned in E coli strain ToplOF' (Grant, et al , 1990) and expressed in Pichia pastoris strain GS 115 (Cregg, et al , 1985), both purchased from Invitrogen (San Diego, CA) Plasmid pASK40 (Skerra, et al , 1991) was used for plasmid construction for E coli expression Yeast expression plasmid pIC-9 (Invitrogen) was used for plasmid construction for yeast expression D Enzymes and Oligonucleotides Restriction enzymes and DNA modifying enzymes were purchased from GIBCO BRL (Gaithersburg, MD) Deoxyribonucleotides were prepared on an automatic DNA synthesizer Model 380A (Perkm-Elmer Applied Biosystems Division. Foster City. CA) Isolation of plasmid DNA and routine manipulations were carried out according to standard procedures (Sambrook, et al , 1989. Ausubel, et al , 1988) Dideoxy-sequencing (Maxam and Gilbert, 1977) of the cloned plasmid constructs was performed using a modified polymerase chain reaction (PCR) cycle sequencing protocol (Craxton, 1991 ) employing biotinalated primers and chemiluminescent detection (Amersham, Arlington Heights, IL) E SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 15% acrylamide gels in a mini-gel apparatus ("MINI-PROTEIN II' Dual Slab Cell, Bio-Rad Laboratories, Hercules CA) as described by Laemmli, et al ( 1973) Gels were stained with Coomassie blue (R-250, Bio-Rad) F Western Blot Proteins on the SDS-PAGE gel were transferred to nitrocellulose membrane using the protocol of Towbm, et al (1979). with a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories) The transfer was typically completed after 30 mm under constant current of 300 mA (Model 200/2 0 Power supply, BioRad) Excess binding s es on the membrane were blocked by incubation of the blots with a blocking solution consisting of 50 mM tπs-hydroxy-methyl ammomethane (Tris) HCl, pH 7 5, 150 mM NaCl, 0 05 % (v/v) "NONIDET-P40", 0 25 % (w/v) gelatin and 3% (w/v) BSA at room temperature for 1 hr in an incubator shaker (model G25 Gyroshaker, New Brunswick Scientific, New Jersey, USA) set at 100 φm The membrane was washed twice at room temperature with 10 mM Tris-HCl buffer pH 7 5 containing 0 1 % "TWEEN-20" and 0 05 % (w/v) BSA (TTBS) G Ligand Blot Proteins resolved on an SDS-PAGE gel were transferred to nitrocellulose membrane as described above The proteins on the membrane were denatured by soaking the membrane for 1 hr in PBS-ED buffer (20 mM potassium phosphate, pH 7 9. 200 mM KCl 1 mM EDTA. 1 mM DTT) containing 6 M guanidine HCl The proteins were gradually renatured by four serial 1 1 dilutions of the membrane in PBS-ED and washed free of the residual guanidine HCl in fresh PBS-ED buffer The membrane was blocked for 1 hr in PBS-ED blocking buffer containing 0 05% (v/v) Nonιdet-P40, 0 25 % (w/v) gelatin and 3% (w/v) BSA at room temperature for 1 hr in an incubator shaker The blots were rinsed once with PBS-ED EXAMPLE 1 Chemical Synthesis. Purification and Analysis Peptides were chemically-synthesized by solid-phase peptide synthesis using a benzhydryl amine-hydrochloπde resin on an Applied Biosystems (Foster City, CA) peptide synthesizer Model 430A with conventional V-f-butyloxycarbonyl (t-Boc) chemistry as described previously (Hodges, et al , 1988) The peptides were cleaved from the resin by reaction with hydrofluoric acid (HF, 20 ml/g resin) containing 10% anisole and 2% 1 ,2- ethanedithiol for 1 hour at -5°C to 0°C The crude reduced peptides were purified by reversed-phase high performance liquid chromatography (RPC) and a "SYNCHROPAK" RP-P semi-preparative C, 8 column (250 x 10 mm inner diameter. 6 5 μm particle size, 300 Λ pore size, SynChrom, Lafayette, IN) with a linear AB gradient of 0 5 % B/min and 2 ml/min, where solvent A was 0 05 % trifluoroacetic acid (TFA) in water and solvent B was 0 05 % TFA in acetonitrile The amino acid composition and mass analysis were consistent with the designed sequence For amino acid analysis, purified peptides were hydrolyzed in 6 N HCl containing 0 1 % phenol at 100°C for 24 hours or 1 hour at 160°C in evacuated sealed tubes Amino acid analysis was performed on a Beckman model 6300 amino acid analyzer (Beckman, San Ramon, CA) The correct primary ion molecular weights of the reduced peptides were confirmed by plasma desoφtion time of flight mass spectroscopy on a BIOION-20 Nordic (Uppsala, Sweden) EXAMPLE 2 Circular Dichroism Measurements Circular dichroism (CD) spectra were recorded at 20°C on a Jasco J-500C spectropolaπmeter (Jasco, Easton, MD) equipped with a Jasco DP-500N data processor and a Lauda (model RMS) water bath (Brinkmann Instruments. Rexdale. Ontario, Canada) for control of the temperature of the cuvette. Constant N, flushing was employed. The instrument was routinely calibrated with an aqueous solution of recrystallized d- \0-( + )- camphorsulfonic acid at 290 nm. Molar ellipticity at 200 nm is reported as mean residue molar ellipticity ([t?] 22 o. deg»cnr»dmol ') and calculated from the equation: W = [Θ , x mrw/10 x / x c [θ]^ is the ellipticity measured in degrees, mrw is the mean residue molecular weight (molecular weight of the peptide divided by the number of amino acid residues), c is the peptide concentration in grams per milliliter, and / is the optical path length of the cell in centimeters. CD spectra were the average of four scans obtained by collecting data at 0.1- nm intervals from 250 to 190 nm. Peptide concentrations were determined by amino acid analysis. The pH was measured at room temperature. EXAMPLE 3 Heterodimer vs. Homodimer Formation Peptides 0993 (SEQ ID NO:26) and 0994 (SEQ ID NO:27) were synthesized as described in Example 1. CD spectra of peptide mixtures of different ratios of the first heterodimer-subunit peptide 0993 (SEQ ID NO: 26) and the second heterodimer-subunit peptide 0994 (SEQ ID NO:27) were measured as described in Example 2, to determine the degree of heterodimer vs. homodimer formation. The peptides were suspended in a solution containing 0. 1 M KCl and 50 mM potassium phosphate buffer, pH 7 at 20°C (reaction buffer). The total peptide concentration (sum of 0993 and 0994 concentrations) was 20 μM. The data show that as the ratio of the peptides was changed from 0: 100 to 50:50, the conformation of the peptide mixture changed from a random coil structure to an α- helical structure. An equimolar mixture of the 0993 and 0994 peptides displayed the double minima at 220 and 208 nm with -31 ,760 deg«cπr«dmol " ' of mean residue ellipticity at 220 nm, which corresponds to - 100% α-helical structure (Hodges, et al. , 1990), suggesting that the interhelical ionic repulsions which destabilize the homo-stranded coiled-coil provide a driving force for the formation of the hetero-stranded coiled-coil. These results indicate that the mixture of peptides 0993 and 0994 forms a hetero- stranded coiled-coil EXAMPLE 4 Linking Peptides to Heterodimer Scaffold bv Alkylation of Thiol Groups Conjugation to peptide sulphydryl groups was carried out at ambient temperature in 50 mM NHjOAc and 8 M urea at pH 8. Bromo- or iodacetylated peptides were dissolved in buffer (0.987 μM, 2 ml) and peptide 0994 (SEQ ID NO:27) was added to a final concentration of 0. 165 μM (2 ml). The reaction mixture remained clear and was allowed to react at ambient temperature for 22 hours, at which time it was acidified by the careful addition of TFA (pH 2) and lyophilized. A. Coniugate Purification and Identification The reaction mixture (2 ml) was applied directly to a Synchropak RP-8 semi-prep column (250 mm x 10 mm I.D.; Synchrom Inc. , Lafayette, IN). The conjugate was easily separated from unreacted peptide using gradient elution (2 % B/minute over 30 minutes. Solvent A: 0.05 % TFA/H 2 0; Solvent B: 0.05 % TFA/acetonitrile) The isolated conjugate was lyophilized and redissolved in HPLC grade water (200 μl) which was then applied to a Mono-S strong cation exchange column (Pharmacia, Uppsala. Sweden) for further purification. The gradient employed during this purification step was a 1 % B/minute gradient (Solvent A: 5 mM NaH : PO 4 /20% acetonitrile. pH 5. Solvent B: 5 mM NaH,PO 4 /20% acetonitrile, 1 M NaCl, pH 5). The isolated conjugate was then desalted using a reversed-phase column and a standard 2 % B gradient (vide supra). In this way, pure conjugate was obtained which was shown through mass spectrometric analysis to be the desired product (MW calc: 7432.0, Found, 7432.4). EXAMPLE 5 Recombinant Coil Gene Synthesis The E-coil gene was synthesized as follows. A DNA sequence was constructed from back translation of the de novo designed coil protein sequence using a codon bias based the on highly expressed Pichia pastoris genes (Koutz, et al. , 1989). To avoid creation of excess restriction sites, four overlapping oligonucleotides were synthesized and purified by electrocution on a 12% polyacrylamide gel containing 7M urea. The coil DNA fragments were synthesized using Klenow fragment of E. coli DNA Polymerase I as shown in Figs. 5A through 5G. Oligonucleotides 1 (SEQ ID NO:6) and 2 (SEQ ID NO:7) were annealed together by their complementary sequences and the remaining complementary strands were synthesized by Klenow fragment as illustrated in Figures 5 A and 5B. An identical protocol was carried out for oligonucleotides 3 (SEQ ID NO:8) and 4 (SEQ ID NO:9) (Figs. 5D and 5E). The double stranded fragments were then digested with restriction enzyme Alw21 I to create sticky ends at one end of each fragment (Figs. 5C and 5F), and the two fragments were ligated with T4 DNA Ligase to generate a double-stranded fragment (Fig. 5G) of 177 base pairs. The fragment was excised, electroeluted from the gel slice, and digested simultaneously with EcoRI and BamHl to produce a 166bp fragment suitable for cloning into a plasmid vector. EXAMPLE 6 Expression of Coil Constructs in E. coli Expression vector pASK40 was modified at its polycloning site to produce plasmid pRLD. pASK40 was digested with EcoRI and the resulting 5' protruding ends were blunted with Mung-Bean Nuclease. The blunted plasmid was then digested with Hindl and religated with a synthetic 43 bp annealed deoxyribonucleotide fragment formed by annealing oligonucleotides 5 (SEQ ID NO: 10) and 6 (SEQ ID NO: 1 1 ). This modification, which was verified by double stranded DNA sequencing, was designed to shift the reading frame and make pASK40's cloning site more consistent with that of Pichia pastoris vectors. The synthesized E-coil gene was double digested with EcoRI and BamHl as described above and force-cloned into pRLD digested with EcoRI and Bglll to produce the E. coli E-coil vector pRLD-E (Figure 5H). The ligated plasmids were transformed into E. coli expression strain JM83 by heat-shock treatment (Yanisch-Perron, et al., 1985) and plated on selective LB plates supplemented with carbenicillin (100 μg/ml). Insertion of the coil gene was verified by restriction digest analysis and subsequent DNA sequencing of minipreped plasmid DNA of successful transformants. E. coli cells harboring pRLD-E were grown at 25 °C with shaking in LB medium containing 100 μg/μl of carbenicillin to an A 550 of 0.5. Expression was induced by adding isopropylthiogalactoside (IPTG) to a final concentration of ImM and incubating for 3 hours at 25 °C with shaking. Expressed protein was obtained by a modified osmotic shock treatment (Ausubel, et al , 1988) Cells were collected at 4000 xg for 10 minutes at room temperature and resuspended in 100 mM Tπs-Cl pH 8 0 containing 5mM EDTA and 20% sucrose (TES buffer) at 80ml per gram wet weight The cells were shaken at 200φm at room temperature for 10 minutes The suspension was then centrifuged again and the pellet resuspended in 5 mM ice-cold MgS0 4 (80 ml per gram wet weight) This was shaken for 30 minutes on ice and subsequently centrifuged at 8000 xg at 4°C for 10 minutes The supernatant constituting the peπplasmic fraction was further purified using the coil affinity column, as described in Example 8, below EXAMPLE 7 Expression of Coil Constructs in Yeast The E-coil gene described above was used as a template for the polymerase chain reaction (PCR, Saiki, et al , 1988, Mul s. 1987, Mulhs, et al , 1987) to generate an E-coil gene with a 5' EcoRI sue and 3' NotI site Synthetic Ecopπ (SEQ ID NO 12) and Notpπ (SEQ ID NO 13) oligonucleotides were used as primers at an annealing temperature of 50°C for 30 cycles The PCR amplification product was cloned into plasmid pIC9 (Invitrogen, San Diego, CA), which contained the alpha mating factor leader sequence for secretion into the medium, generating Pichia E-coil vector p9CE Successful ly- gated plasmids were transformed into E coli cloning strain TOP10 F' and plated on LB/Carbenicil n ( 100 μg/ml) plates Plasmid DNA was prepared from selected colonies and checked for the correct size of insert by digestion with restriction enzymes and confirmed by DNA sequence analysis Verified yeast plasmids were transformed into Pichia pastoris strain GS 1 15 as described (Cregg, et al , 1985) GS1 15 was cultured in YPD (1 % peptone, 2% yeast extract, 2% dextrose) media (Romanos, et al , 1991) at 30°C with shaking to an A^ of 0.25 and harvested by centrifugation The cell pellet underwent a series of washes with dH 2 0 and IM aqueous sorbitol, followed by IM aqueous sorbitol containing 25mM EDTA and 100 mM dithiothreitol (SED buffer) and again with IM sorbitol The final pellet was resuspended in IM aqueous sorbitol containing 10 mM sodium citrate buffer, pH 5 8, and ImM EDTA (SCE buffer) and treated with zymolase at a final concentration of 0 3mg/ml Spheroplasting was carried out at 30°C and monitored by treating aliquots taken at various time points with 5 % SDS and checking their absorbance at 800nm At 70% spheroplasting, the cells were harvested by centrifugation and washed with IM aqueous sorbitol. followed with a wash in I M aqueous sorbitol containing 10 mM Tris-HCl, pH 7 5 and 10 mM CaCl : (CaS buffer) The spheroplasts were resuspended in CaS and used immediately for DNA transformation. In preparation for transformation, 10 μg of plasmid p9CE DNA was digested with restriction enzyme Bglll to generate two linear fragments, confirmed by electrophoresis on a 0 7% agarose gel. Newly-prepared spheroplasts were incubated with digested DNA for 10 minutes at room temperature. A fresh solution of 20% PEG with 10 mM CaCl : and 10 mM Tris-HCl, pH 7.5 (PEG/CaT) was added and incubation continued for another 10 minutes. Cells were harvested at 750 xg at room temperature and the supernatant aspirated off The pellet was resuspended in SOS media (1 M sorbitol, 0.3X YPD media, 10 mM CaCU) and the cells were allowed to recover for 20 min before addition of IM aqueous sorbitol and plating on selective RDB plates (1 M Sorbitol, 1 % dextrose, 1.34% yeast nitrogen base, 4 x 10-5 % biotin and 0.005 % amino acids without histidine). Recombined clones were identified by growth in the absence of histidine, and displacement of the chromosomal AOX1 gene was distinguished by growth on selective minimal methanol media ( 1.34% yeast nitrogen base, 4 x 10-5% biotin, 0.5 % methanol). Clones that had undergone successful integration of the linerarized plasmid fragment were identified by PCR screening (Clare, et al. , 1991) using vector-specific primers (SEQ ID NO: 14, SEQ ID NO: 15). Recombinant yeast clones exhibiting a methanol sensitive phenotype (MetS) were cultured in BMGY media (peptone, yeast extract, yeast nitrogen base, biotin. glycerol) to an A,™ of 1.0. The carbon and energy source was subsequently changed to methanol (BMMY media) to allow for induction using the AOX 1 promoter (Ellis, et al. , 1985). After 72 hours of induction, the culture supernatant was harvested by centrifugation at 4000 xg and filtered through a 0.2 μm filter in preparation for coil affinity purification, described in Example 8, below. EXAMPLE 8 Purification of Proteins on Selective Dimerization Affinity Matrix Affinity Column This section describes preparation and use of an affinity matrix designed for purification of polypeptides containing a coil peptide. The affinity purification procedure is based on selective dimerization of a heterodimer-subunit peptide immobilized on a solid phase to a proteins or peptides containing a complementary heterodimer subunit. Particularly , polypeptides selected for purification are fusion peptides designed and synthesized to contain a heterodimer-subunit peptide complementary to the immobilized peptide Generally, to prepare a selective dimerization affinity matrix, one of a pair of complementary peptide subunits is synthesized as a linear peptide according to methods known in the art purified, then conjugated to a solid matrix through one of the residues present in the peptide Preferably, a cysteine residue will be available for conjugation of the peptide The sections that follow describe preparation of an affinity matrix having conjugated thereto a heterodimer-subunit peptide Also detailed below are conditions used for binding the complementary heterodimer-subunit peptide to the affinity matrix of and elution of the complementary subunit from the column A Preparation of Selective Dimerization Affinity Matrix 1 Preparation of Peptides Complementary peptides were designed as described above to form a stable α-helical coiled-coil heterodimer For conjugation to the solid phase, one of the peptide subunits was synthesized to include a spacing group, shown as the C-terminal 5 ammo acids in SEQ ID NO 27, and to have a C-terminal cysteme-amide for convenience of chemical coupling to the matrix The following pair of complementary peptides was designed and prepared as follows SEQ ID NO 26 (Peptide 0993) Ac-EVSALEKEVSALEKEVSALEKEVSALEKEVSALEK-Amide SEQ ID NO 27 (Peptide 0994) Ac-KVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGGGnLC-Amide gabcdefgabcdefgabcdefgabcdefgabcdef The peptides were synthesized on a solid phase peptide synthesizer (430A Applied Biosystems Inc , Foster City, CA) using standard t-BOC chemistry Peptide was cleaved from the synthesis resin by treatment with hydrogen fluoride Purification of each peptide was carried out using reversed phase high performance liquid chromatography (RP-HPLC) 2 Coupling of Peptide to Affinity Matrix Control pore aminopropyl glass resin (220 mg (22 μmol), Sigma, G-4643, average pore 500 Angstrom, 200-400 mesh, amine content 1 l ϋumol/g glass) was washed twice with 5 0 ml each of DMF DCM and DMF The resm was neutralized with 5 0 ml of a DIEA'DCM solution (5 % v/v) for 5 0 minutes and then drained The resin was next stirred for 25 minutes in a solution of bromoacetic acid dissolved in DCC/DCM solution, prepared by dissolving 138 mg ( 1 0 mmol) of Bromoacetic acid (Aldrich Fine Chemicals Catalog #25,935-7) in 2 0 ml of a 0 5 M DCC/DCM solution stirring for fifteen minutes and filtering to remove insoluble DCU The resin was again drained and washed three times each with 5 0 ml DCM, DMF, MeOH, DCM The resultmg activated glass resin was then dried Peptide 0994 (SEQ ID NO 27, 26 mg, 6 15 μmol) was dissolved in 3 0 ml of a 100 mM NH 4 HC0 3 solution, pH 8 0 This solution was added to the activated glass resin beads The peptide resin solution was stirred for 1 5 hours, after which the resin was drained and washed with 5 0 ml H 2 0 Remaining unreacted bromoacetyl groups were capped by adding 5 0 ml of a 1 % v/v solution of /3-mercaptoethanol in 100 mM NH 4 HC0 3 and the resin mixture stirred for thirty minutes The resin was then drained and washed 5x with H 2 0, DMF, and methanol then air dried for storage Coupling was verified by qualitative ninhydrin test B Preparation of Affinity Column An RP-HPLC guard column (5 mmx20 mm) was dry-packed with the prepared dimerization chromatography resin The column was then washed for 10 minutes at a flow rate of 0 2 ml/mm with the following solutions, H 2 0. 50% acetonιtrιle-H,0. 5 0 M GndHCl in 50 mM potassium phosphate pH 7 8. and H 0 This extensive washing was carried out in order to remove from the column any non-specific noncovalently attached peptide C Chromatography of Heterodimer-Subunit on Selective Dimerization Affinity Matrix An affinity column prepared as detailed in Part B, above was equilibrated in 100% H 2 0 at a flow rate of 0 2 ml/min A 50 μl sample of peptide 0993 (SEQ ID NO 26, 1 mg/ml in 10 mM phosphate buffer, pH 6 5) was injected and eluate fractions were collected for analysis by RP-HPLC RP-HPLC was carried out on a C-8 Zorbax analytical column equilibrated with 0 05 % TFA in H 2 0, and eluted with a gradient of 2% /min solution B (0 05 % TFA acetonitrile) at a flow rate of lml/min and detection at 210 nm Following injection of sample onto the affinity column, the column was washed with a solution of 60% acetonιtπle/H 0 tor 5 0 mm and the eluate during the wash step was collected for analysis on RP-HPLC The peptide was subsequently eluted from the affinity column with a solution of 5 0 M GndHCl in 50mM potassium phosphate pH 7 8, followed by several volumes of H 2 0 Figure 7A shows a chromatogram of peptide 0993 eluting from an RP-HPLC column at about 25 minutes Figure 7B shows a chromatogram of the injection flow through fraction analyzed by RP-HPLC under the conditions described above, after injecting peptide 0993 onto the selective dimerization affinity column described in Part C, above Absence of a peak at 25 min shows that substantially all loaded peptide bound to the column Figure 8 shows a chromatogram of the 60% acetonitrile wash fraction of the affinity column The presence of only a minor peptide peak at the peptide 0993 position indicates that the peptide was not eluted by the washing procedure Figure 9 shows a chromatogram of the GndHCl elution fraction The major peak eluting at approximately 25 minutes is peptide 0993, indicating that 0 5 M GndHCl is effective to elute this fraction D Conditions for Removal of non-specific Peptides from Selective Dimerization Affinity Column Studies were carried out in support of the present invention to determine conditions for removing non-specific binding of charged peptides from the affinity column The following peptides were used in a test mixture Peptide Test Mixture Peptide Seguence Net charge at pH 6 0 SEQ ID NO 28 (Actin 1 -28) Ac-DEDETTALVADNGSGLUKAGFAGADAPR-Amide -4 SEQ ID NO 29 (Anion Exchange Standard) Ac-EYAAEAAEGLE-Amide -4 SEQ ID NO 30 (Tnl 104-115) Ac -GKFKRPPLRRVR-Amide +6 To test for non-specific binding, the affinity column was equilibrated in 10 mM phosphate pH 6 5 at a flow rate of 0 2ml/mm A 100 μl sample of the peptide test mixture ( 1 mg/ml Actin 1-28. 1 mg/ml Anion exchange std , 2 mg/ml Tnl 104-1 15 in 10 mM phosphate buffer, pH 6 5) was injected and the eluate during injection collected for analysis on RP-HPLC Figure 10 shows a chromatogram of the peptide mixture, run on the RP-HPLC column prior to loading on the affinity column The conditions for running this column were identical to those detailed m Part D, above Indicated in the figure are 3 major peaks representing the three above- referenced peptides in the mixture The affinity column was then washed with a solution of 0 2 M KCl/ 10 mM phosphate for 5 0 mm and the eluate during the wash step collected for analysis on RP-HPLC Figure 1 1 shows a chromatogram of the eluate fraction The single major peak eluting at about 12 minutes indicates that Tnl 104-1 15 peptide (SEQ ID NO 28) having a net positive ( + 6) charge was not retained by the column, while both the negatively charged proteins were retained The affinity column was then washed with a solution of 0 5 M KCl/ 10 mM phosphate for 5 0 mm and the eluate during the wash step collected for analysis on RP-HPLC Figure 12 shows no detectable peptide peaks eluting at positions corresponding to any of the components of the peptide mixture The affinity column was then washed with a solution of 1 0 M KCl/ 10 mM phosphate for 5 0 min and the eluate during the wash step collected for analysis on RP-HPLC The chromatogram depicted in Figure 13 shows that both the anion exchange standard peptide (SEQ ID NO 29) and the actin 1 -28 peptide (SEQ ID NO 30) were eluted Subsequent washes were carried out, using (a) 1 0 M KCl, and (b) a solution of 5 0 M GndHCl in 50mM K 2 P0 4 pH 7 8 These fractions were collected for RP-HPLC analysis as described above Figures 14 and 15 show that no additional test peptides were eluted from the column under these conditions E Purification of Recombinant PAK pilin/E-coil peptide This section describes purification of a recombinant PAK pilin/E-coil fusion protein The fusion protein was produced as detailed in Example 9 1 Preparation of Peptide Affinity Matrix One gram (1 10 μmol) of aminopropyl control pore glass resin (Sigma , Cat# G-4643) was washed 2x with 15 0 ml of DMF, DCM and DMF The resm was neutralized with 15 0 ml of a 5 % v/v DIEA/DCM solution for 5 0 minutes and then drained Two ml of a 0 5 M dicyclohexylcarbodnmide/DCM solution were added to 138 mg (1 0 mmol) of a 0 5 M dicyclohexylcarbodπmide/DCM solution, and the mixture was stirred for fifteen minutes The mixture was then filtered and the filtrate added to the prepared glass resin beads The final volume of the mixture was brought up to 20 ml with dichloromethane The mixture was stirred for 25 minutes and then drained The resin was washed 3x with 15 0 ml DCM, methanol, and DCM, then dried A qualitative ninhydrin test was performed to verify coupling One hundred milligrams (23 7 μmol) of peptide 0994 was dissolved in 30 0 ml of a 100 mM NH 4 HCO,, pH 8 0 and then added to the glass resin beads The peptide resin solution was stirred for 30 minutes, after which the resin was drained and washed with 5 0 ml H 2 0 Remaining unreacted bromoacetyl groups were capped by adding 20 0 ml of a 1 % v/v solution of 3-mercaptoethanol in 100 mM NH 4 HC0 3 , pH 8 0 to the resm, and the mixture was stirred for fifteen minutes The resin was then drained and washed 5x with H 2 0, 0 1 % TFA H 2 0. Ethanol, Acetone and then dried for storage A small amount of resin was removed for amino acid analysis and theoretical capacity determination 2 Preparation of Affinity Column Two columns were prepared for affinity purification, i) a stainless steel RP-HPLC guard column (5 mm x 20 mm), and n) a stainless steel RP-HPLC column (10 mm x 200 mm) Each column was dry-packed to the top with the prepared dimerization chromatography resin After packing, each column was washed at a flow rate of 0 5 ml/mm with the following solutions, H : 0, 5 0 M GndHCl in 50 mM potassium phosphate pH 7 0. 50% acetonιtπle/H 2 0 with 0 1 % TFA, and H,0 3 Purification of Recombinant Peptides The columns were first conditioned in 10 mM phosphate buffer, pH 6 0 Filtered peπplasmic fractions or whole cell extracts were loaded at a flow rate of 0 2-0 5 ml/min After loading, the columns were washed with 5 0 ml (small column) or 20 ml (large column) of 0 5 M KCl in 10 mM phosphate buffer pH 6 0 followed by 80% acetonitrile in 10 mM phosphate buffer pH 6 0 (v/v) Final elution of bound peptides was carried out with a wash of 50% acetonιtπle/H 2 0 with O l %TFA (v/v/v) Presence of recombinant protein in eluted fractions was assessed by RP-HPLC (Zorbax C-8 analytical column (10 mm x 250 mm), at a flow rate of 1 0 ml/min with a linear AB gradient of 1 0% B/min from 0% to 60% B, where solvent A was 0 05% trifluoroacetic acid (TFA) in water and solvent B was 0 05 % TFA in acetonitrile The total run time was 60 0 min Figure 17 shows RP-HPLC chromatograms ot load samples and tractions eluted from the affinity column when the load fraction was a crude penplasmic extract containing recombinantly expressed E-coil peptide HPLC runs are indicated as follows a. pre- elution, b. crude penplasmic extract, c, break-through of penplasmic extract, d, 0 5 M KCl wash, e, 80% acetonitrile wash, f, first elution wash, g. second elution wash, h, first elution wash after re-passage of break-through, followed by washes Figure 18 shows RP-HPLC chromatograms of purification of recombinantly expressed PAK-pili-E-coil peptide from crude penplasmic extract All HPLC runs were carried out as described above HPLC runs are indicated as follows a, pre-elution, b. crude penplasmic extract, c, break-through of penplasmic extract, d, 0 5 M KCl wash, e, 80% acetonitrile wash, f first elution wash, g. second elution wash EXAMPLE 9 PAK Pih Antigen/E-Coil Fusion Two complementary oligonucleotides (oligonucleotides SEQ ID NO 16 and SEQ ID NO 17) encoding the 17 amino acid pilus epithelial binding domain (SEQ ID NO 5) of the PAK pili ( 128- 144, Lee et al , 1994) were synthesized Equal molar ratios of the oligonucleotides were dissolved in sterile dH 2 0. mixed together, heated to 80°C for 5 mm and allowed to anneal by slow cooling to room temperature The annealed oligonucleotides were electrophoresed on a 4% agarose gel and a 54 bp fragment was excised and eluted from the gel slice by a modified freeze-squeeze method (Sambrook, et al . 1989) Briefly , the excised band was placed in a spin filter column (BioRad. Richmond. CA) and placed on liquid nitrogen for 5 minutes The filter was then placed into an eppendorf tube and spun at top speed ( ~ 14.000 φm) in a microfuge Following ethanol precipitation, the eluate was treated with T4 polynucleotide kinase at 37°C for 30 minutes and the enzyme inactivated at 65°C for 15 min The phosphorylated fragment was then cloned into shrimp alkaline phosphatase-treated, EcoRl- digested pRLDE and transformed into E coli strain JM83 to produce pRLDE-PAK clones on LB/Carbenicillin plates Orientation and sequence of the insert was verified by restriction analysis and sequencing of mi preped DNA plasmid Expression and purification of the PAK/E-coil fusion protein was carried out as previously described for the E-coil protein EXAMPLE 10 Green Fluorescence Protein/E-Coil Fusion A Green Fluorescent Protein (GFP) cDNA clone (Prasher, et al , 1992) was purchased from ClonTech (Palo Alto, CA) GFP-specific PCR primers (oligonucleotides SEQ ID NO 18 and SEQ ID NO 19) were constructed with flanking EcoRI restπction sues and PCR of the GFP gene was carried out under standard conditions (Saiki, et al . 1988) The PCR product was run on a 1 % agarose gel and the 700bp amplification product was excised and eluted from the gel as described above Plasmid pRLDE-GFP was constructed by digesting the eluted DNA fragment with EcoRI and Iigating the DNA into shrimp alkaline phosphotase-treated, £cσ/?7-dιgested pRLD-E pRLDE-GFP was then transformed into E coli strain TOP 10 F' Orientation of the gene was determined by direct detection of fluorescent colonies on LB/CA/IPTG transformation plates ( 100 μg/μl carbenicillin, 0 8mM IPTG) The sequence of the gene was confirmed by restπction analysis and sequencing Expression and purification of the protein was earned out as previously described EXAMPLE 1 1 Western and Ligand Blot Analyses of PAK/E-coil Fusion Crude penplasmic prep from E coli JM 83 cells containing expressed PAK 17 mer peptide tagged with E-coil. and PAK 17/E-coιl purified by the K-coil affinity column were fractionated by SDS-PAGE at a constant voltage of 200V with a power supply model 1420A (BioRad Laboratories) Figure 16A shows an image of the gel stained with Coomassie blue The lanes are as follows lane 1 crude PAK( 128- 144)/E-coιl penplasmic prep, lane 2 PAK ( 128- 144)/E-coιl purified by K-coil affinity column, lane 3 rainbow marker (2-45 kda) The gel was blotted and subjected to Western Blot analysis as described above Monoclonal antibody PK99H, raised against the Pseudomonas aeruginosa strain K pi (PAK pili), was diluted with TTBS (1 500). and incubated with the blots for one hour at room temperature with gentle shaking The blots were then washed three times with TTBS, and a goat anti-mouse IgG(H + L)-alkahne phosphatase conjugate (Jackson Laboratories) diluted 1 10.000 with TTBS was incubated with the blots as described above The blots were washed 3 times with TTBS followed by a final wash with Tπs-buffered saline Antibody binding was visualized by the addition of alkaline phosphatase substrates (nitro blue tetrazolium chloπde and 5-bromo-4-chloro-3-ιndoly 1 phosphate dissolved in 100 mM Tris-HCl pH 9 containing 100 mM NaCl and 5 mM MgCK) Color development was stopped by rinsing the nitrocellulose strips with deionized water Ligand-blots were performed to evaluate the ability of biotm-labelled (bt) K-coil to act as a probe A gel as shown in Figure 16A was blotted as described above, and biotin-labelled K-coil ( 1 mg/ml) was diluted 1 500 in PBS-ED blocking buffer and incubated with the blots at room temperature for 1 hr with gentle agitation The membranes were washed three times. 10 minutes per wash Strepavidin-alkahne phosphatase conjugate (Jackson ImmunoResearch Laboratory) was diluted ( 1 2500, vol/vol) with PBS-ED blocking buffer and incubated with the blots at room temperature for 1 hr with gentle agitation The blots were washed 3 times with PBS-ED followed by a final wash with substrate buffer (100 mM Tris-HCl pH 9 5. 100 mM NaCl and 5 mM MgC12) K-coil binding was visualized by the addition of nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-ιndolyl phosphate (NBT/BCIP) in substrate buffer Color development was stopped by rinsing the nitrocellulose strips with deionized water The results of the Western and ligand Blot analyses are shown in Figure 16B Lanes 2-3 and 8-9 were probed using the ligand blot technique, while lanes 5-6 and 1 1 - 12 were probed using the Western blot technique Lanes 1 , 4. 7 and 10 contained size markers (Rainbow marker. 2-45 kDa), lane 2 bt K-coil binding to crude PAK( 128- 144)/E-coιl penplasmic prep, lane 3 bt K-coil binding to PAK( 128- 144)/E-coιl purified by K-coil affinity column, lane 5 monoclonal antibody , PK99H. binding to crude pak ( 128-144)/E- coil penplasmic prep, lane 6. monoclonal antibody , PK99H, binding to purified PAK ( 128- 144)/E-coιl by K-coil affinity column, lane 8. bt K-coil binding to E-coil purified by K-coil affinity column, lane 9. Bt-K-coil binding to PAK pih, lane 1 1. monoclonal antibody. PK99H, binding to E-coil purified by K-coil affinity column, lane 12, normal mouse IgG binding to PAK pi EXAMPLE 12 Isolation of PAK/E-coil Fusion Using K-coil Affinity Column A Isotope ( l5 N) Labelling A single colony of E coli strain JM83 harboring the recombinant expression plasmids (pRLDE-PAK) was inoculated in 5 ml LB medium contain 100 μg/ml carbenicillin (Sigma) and grown at 37 °C overnight The overnight culture (2 ml) was sub-inoculated in 200 ml of mini-medium (0 67- NdH ,P0 , 0.3 % K,HP0 . 0 05 % NaCl. 4 mM MgS0 4 , 2 μM FeS0 4 . 0 1 % "NH 4 C1 (Cambridge Isotope Laboratories. Woburn. MA) and 1 % D-glucose, pH 7.2) The lnducer, IPTG, was added to culture to final concentration ot 1 mM once OD N , reached 0 8 to 1 0 The cultures were grown at 37 °C for another 6 hr and applied for further purification B Preparation of E coli Penplasmic Prep The induced gene-expression cultures were harvested by centrifugation (4000 xg, 10 min). The pellets were resuspended in 100 ml of TES buffer containing 100 mM Tris, 10 mM EDTA and 20% sucrose and incubated at room temperature for 10 min with gentle agitation The cells were collected by centrifuging (7000 xg. 10 mm) and resuspended in 5 mM MgS0 4 The suspension was incubated in ice-cool bath for 30 min with agitation The supernatant which contained the penplasmic proteins was collected by centrifugation (5000 xg, 10 min) and stored at -20°C or freeze-dried C HPLC-Affinitv Column for Purification of PAK/E-coil The freeze-dried expressed crude penplasmic preps were dissolved in 20 ml of 10 mM phosphate buffer (pH 6). The dissolved sample (5 ml) was loaded on an affinity column conjugated with K-coil peptide after the column was washed and equilibrated with 10 mM phosphate buffer (pH 6) The constant flow rate of HPLC was 0.2 ml/min The column was washed by injecting 5 ml 0.5 M KCl (pH 6) in phosphate buffer 25 minutes after the sample was loaded. Five ml of 80% acetonitrile in 10 mM phosphate buffer pH 6 5 were injected 25 mm after the 0.5 M KCl washing The PAK/E-coil fusion was eluted with 5 ml of 50% acetonitrile containing 0 1 % TFA in ddH : 0 The identity of the eluted sample was confirmed using reversed phase chromatography and mass spectrometer analysis EXAMPLE 13 Detection Reagent This section describes synthesis and use of a reagent for detecting fusion proteins having a heterodimer-subunit peptide capable of forming an α-helical coiled-coil heterodimer with a complementary peptide in conformance with the invention A Iodinated Reagent A heterodimer-subunit peptide is formed as described above The peptide is then iodinated according to standard procedures known in the art Preferably heterodimer- subunit peptide designed for iodination contains a tvrosme residue however iodination can also be accomplished by use of, e g . a Bolton-Hunter Reagent which preferably attaches to lysine residues For example, lysines occupying position f of the subunit repeat unit may be so derivatized Kits containing Bolton-Hunter reagent are commercially available (e g ICN, Costa Mesa, CA) Another iodination method is the "IODOGEN' method Here, 2 mCi of carπer- free Na' : T 75 μl 0 5M phosphate buffer pH 7 4 and 20 μl of 1 μg/μl peptide are added to a polypropylene test tube coated with 10 μg "IODOGEN' The tube is agitated for 8 minutes and the solution is chromatographed by HPLC through a 10 x 0 46 cm C-8 reversed phase column with a pore size of 300 A (Brownlee Labs Santa Clara CA) The sample material is eluted with a gradient from 0 1 % trifluoroacetic acid to 60% acetonitrile in 0 1 % trifluoroacetic acid The major peak of active radio-iodinated peptide is detected by gamma-detection of fractions and is used as a reporter molecule in the method of the invention B Enzyme-linked Reagent An enzyme linked reagent can be made according to methods well known in the art In one such method. a gene for the desired reporter enzyme, such as alkaline phosphatase or luciferase. is fused to a gene for the heterodimer subunit. and the expressed fusion protein purified by affinity chromatography on a complementary heterodimer-subunit peptide affinity column, according to the methods described above In another method, the heterodimer-subunit peptide is chemically linked to the reporter enzyme For example, horseradish peroxidase (HRP) can be conjugated to a cysteine residue present in the peptide subunit, preferably placed in the f heptad position of the coil peptide, through a disulfide bond In one method, HRP (6mg/ml in 0 IM sodium phosphate buffer, pH 7) is incubated for 0 5-1 hour at 30°C with 0 3-0 7 mg of cross- linking reagent (N-succimmidyl 4-(N-maleιmιdomethyl)cyclohexane-l -carboxylate (SMCC) or N-succinimidyl 6-maleιmιdohexanoate (SMH)) dissolved m N,N-dιmethylformamιde (10% solution) The reacted HRP is isolated by gel filtration of Sephadex G-25 (equilibrated with 0 IM phosphate, pH 6, Pharmacia, Piscataway , NJ) To couple the reactive HRP with a reduced cysteine-containing heterodimer subunit the two components are mixed at a 1 1 molar ratio and a final concentration ot 0 01-0 15 mM in 0 1 M sodium phosphate buffer. pH 6 containing 2 mM EDTA. at 4°C tor 20 hours or at 30°C for 1 hour The conjugated product can be puπfied on "ULTROGEL" Ac A 44 (Pharmacia Biotech, Piscataway, NJ) The HRP-conjugated heterodimer-subunit peptide acts as a reporter molecule when it is reacted with 4-amιnoantιpyπne as substrate and increased absorbance at 510 nm is measured While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention SEQUENCE LISTING (1) GENERAL INFORMATION (l) APPLICANT PENCE (li) TITLE OF INVENTION. Coiled-Coil Heterodimer Methods and Compositions for the Detection and Purification of Expressed Polypeptides (m) NUMBER OF SEQUENCES: 30 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Dehlinger & Associates (B) STREET: 350 Cambridge Avenue, Suite 250 (C) CITY: Palo Alto (D) STATE: CA (E) COUNTRY: USA (F) ZIP: 94306 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1 0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: PCT/US96/ (B) FILING DATE. 04-OCT-1996 (C) CLASSIFICATION. (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/540,397 (B) FILING DATE: 06-OCT-1995 (vm) ATTORNEY/AGENT INFORMATION: (A) NAME: Sholtz, Charles K. (B) REGISTRATION NUMBER: 38,615 (C) REFERENCE/DOCKET NUMBER: 8900-0109.41 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (415) 324-0880 (B) TELEFAX: (415) 324-0960 (2) INFORMATION FOR SEQ ID NO 1 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 105 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS double (D) TOPOLOGY both (ll) MOLECULE TYPE DNA (ill) HYPOTHETICAL NO (iv) ANTI-SENSE NO (vi) ORIGINAL SOURCE (C) INDIVIDUAL ISOLATE E-coil sequence (ix) FEATURE (A) NAME/KEY CDS (B) LOCATION 1 105 (xi) SEQUENCE DESCRIPTION SEQ ID NO 1 GAG GTA TCC GCT TTA GAG AAA GAA GTT TCT GCT CTC GAA AAA GAG GTC 48 Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu Lys Glu Val 1 5 10 15 AGT GCT CTG GAA AAA GAG GTG TCA GCC TTG GAA AAG GAA GTA TCA GCA 96 Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala 20 25 30 CTT GAG AAG 105 Leu Glu Lys 35 (2) INFORMATION FOR SEQ ID NO 2 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 35 ammo acids (B) TYPE amino acid (D) TOPOLOGY linear (ii) MOLECULE TYPE protein (Xl) SEQUENCE DESCRIPTION SEQ ID NO 2 Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu Lys Glu Val 1 5 10 15 Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala 20 25 30 Leu Glu Lys 35 (2) INFORMATION FOR SEQ ID NO 3 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 105 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS double (D) TOPOLOGY both (ll) MOLECULE TYPE DNA (i l) HYPOTHETICAL NO (iv) ANTI-SENSE: NO (Vl) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: K-coil seguence (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..105 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 : AAG GTA TCC GCT TTA AAA GAG AAA GTT TCT GCT CTG AAA GAA AAG GTC 48 Lys Val Ser Ala Leu Lys Glu Lys Val Ser Ala Leu Lys Glu Lys Val 1 5 10 15 AGT GCT CTG AAG GAG AAG GTG TCA GCC TTG AAG GAA AAG GTT TCA GCA 96 Ser Ala Leu Lys Glu Lys Val Ser Ala Leu Lys Glu Lys Val Ser Ala 20 25 30 CTT AAA GAG 105 Leu Lys Glu 35 (2) INFORMATION FOR SEQ ID NO:4 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 : Lys Val Ser Ala Leu Lys Glu Lys Val Ser Ala Leu Lys Glu Lys Val 1 5 10 15 Ser Ala Leu Lys Glu Lys Val Ser Ala Leu Lys Glu Lys Val Ser Ala 20 25 30 Leu Lys Glu 35 (2) INFORMATION FOR SEQ ID NO: 5 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: PAK peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Lys Cys Thr Ser Asp Gin Asp Glu Gin Phe Ile Pro Lys Gly Cys Ser 1 5 10 15 Lys (2) INFORMATION FOR SEQ ID NO.6 * (l) SEQUENCE CHARACTERISTICS: (A) LENGTH. 49 base pairs (B) TYPE* nucleic acid (C) STRANDEDNESS. single (D) TOPOLOGY linear (ll) MOLECULE TYPE. DNA (ill) HYPOTHETICAL NO (iv) ANTI-SENSE NO (vi) ORIGINAL SOURCE (C) INDIVIDUAL ISOLATE: Oligonucleotide #1 (x ) SEQUENCE DESCRIPTION: SEQ ID NO:6: GGAATTCGGA GGAGGTGGAG GTGGTGGTGG CGAGGTATCC GCTTTAGAG 49 (2) INFORMATION FOR SEQ ID NO: 7 : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 65 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY, linear (ii) MOLECULE TYPE* DNA (in) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Oligonucleotide #2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: . GTCGTATCAA GAGCACTGAC CTCTTTTTCG AGAGCAGAAA CTTCTTTCTC TAAAGCGGAT 60 ACCTC 65 (2) INFORMATION FOR SEQ ID NO:8: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY, linear (ll) MOLECULE TYPE: DNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Oligonucleotide #3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GTCGTATCAA GTGCTCTGGA AAAAGAGGTG TCAGCCTTGG AAAAGGAAGT ATCAGCACTT 60 GAGAAG 66 (2) INFORMATION FOR SEQ ID NO: : (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (il) MOLECULE TYPE: DNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Oligonucleotide #4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CGGGATCCTT ATTAGTGATG GTGGTGATGT CCTCCACCGC CCTTCTCAAG TGCTGATAC 59 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (il) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Oligonucleotide #5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CTCGAGGGTA CCGAATTCCT GCAGAGATCT GCGGCCGCAC A 41 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Oligonucleotide #6 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: AGCTTGTGCG GCCGCAGATC TCTGCAGGAA TTCGGTACCC TCGAG 45 (2) INFORMATION FOR SEQ ID NO: 12: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ll) MOLECULE TYPE: DNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Oligonucleotide #7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGAATTCGGA GGAGGTGG 18 (2) INFORMATION FOR SEQ ID NO:13: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (li) MOLECULE TYPE: DNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO ( i) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Oligonucleotide #8 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TTTTCCTTTT GCGGCCGCTT ATTAGTGATG GTGGTG 36 (2) INFORMATION FOR SEQ ID NO: 14: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (il) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Oligonucleotide #9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14 : GACTGGTTCC AATTGACAAG C 21 ( 2 ) INFORMATION FOR SEQ ID NO * 15 ( l ) SEQUENCE CHARACTERISTI CS : ( A ) LENGTH : 21 base pairs ( B ) TYPE : nucle i c acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Oligonucleotide #10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GCAAATGGCA TTCTGACATC C 21 (2) INFORMATION FOR SEQ ID NO: 16: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ll) MOLECULE TYPE: DNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Oligonucleotide #11 (Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AATTCAAGTG TACTTCTGAC CAAGACGAGC AATTCATCCC TAAGGGTTGT TCCA 54 (2) INFORMATION FOR SEQ ID NO:17: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ll) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Oligonucleotide #12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: AATTTGGAAC AACCCTTAGG GATGAATTGC TCGTCTTGGT CAGAAGTACA CTTG 54 (2) INFORMATION FOR SEQ ID NO.18 (l) SEQUENCE CHARACTERISTICS. (A) LENGTH 31 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (li) MOLECULE TYPE DNA (ill) HYPOTHETICAL NO (iv) ANTI-SENSE NO (vi) ORIGINAL SOURCE (C) INDIVIDUAL ISOLATE: Oligonucleotide #13 (xi) SEQUENCE DESCRIPTION: SEQ ID NO-18: GGAATTCATG AGTAAAGGAG AAGAACTTTT C 31 (2) INFORMATION FOR SEQ ID NO.19: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH 30 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS* single (D) TOPOLOGY linear (ll) MOLECULE TYPE DNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE. NO (vi) ORIGINAL SOURCE (C) INDIVIDUAL ISOLATE: Oligonucleotide #14 (Xl) SEQUENCE DESCRIPTION: SEQ ID NO:19* GGAATTCTTT GTATAGTTCA TCCATGCCAT 30 (2) INFORMATION FOR SEQ ID NO:20. (l) SEQUENCE CHARACTERISTICS. (A) LENGTH 15 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS unknown (D) TOPOLOGY unknown (ll) MOLECULE TYPE DNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: DNA encoding enterotoxin cleavage site (ix) FEATURE: (A) NAME/KEY CDS (B) LOCATION. 1..15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO 20 GAC GAT GAC GAT AAG 15 Asp Asp Asp Asp Lys 1 5 (2) INFORMATION FOR SEQ ID NO.21 (l) SEQUENCE CHARACTERISTICS. (A) LENGTH: 5 a ino acids (B) TYPE, amino acid (D) TOPOLOGY, linear (ii) MOLECULE TYPE- protein (vi) ORIGINAL SOURCE* (C) INDIVIDUAL ISOLATE Enterotoxin cleavage site (xi) SEQUENCE DESCRIPTION. SEQ ID NO:21: Asp Asp Asp Asp Lys 1 5 (2) INFORMATION FOR SEQ ID NO.22: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS* unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE DNA encoding factor Xa cleavage site (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..12 (Xl) SEQUENCE DESCRIPTION: SEQ ID NO:22 ATC GAA GGT CGT Ile Glu Gly Arg 1 (2) INFORMATION FOR SEQ ID NO:23: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (n) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Factor Xa cleavage site (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23 Ile Glu Gly Arg 1 (2) INFORMATION FOR SEQ ID NO:24 (l) SEQUENCE CHARACTERISTICS (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ll) MOLECULE TYPE: DNA (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE. NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: DNA encoding thrombin cleavage site (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..18 (Xl) SEQUENCE DESCRIPTION: SEQ ID NO:24: CTG GTT CCG CGT GGA TCC 18 Leu Val Pro Arg Gly Ser 1 5 (2) INFORMATION FOR SEQ ID NO:25- (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (il) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE. Thrombin cleavage site (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: Leu Val Pro Arg Gly Ser 1 5 (2) INFORMATION FOR SEQ ID NO:26: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 ammo acids (B) TYPE: amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: 0993 peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu Lys Glu Val 1 5 10 15 Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala 20 25 30 Leu Glu Lys 35 (2) INFORMATION FOR SEQ ID NO: 27: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (li) MOLECULE TYPE: peptide (lli) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: 0994 peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 39..39 (D) OTHER INFORMATION: /label= nLeu /note= "where "Leu" is norleucine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Lys Val Ser Ala Leu Lys Glu Lys Val Ser Ala Leu Lys Glu Lys Val 1 5 10 15 Ser Ala Leu Lys Glu Lys Val Ser Ala Leu Lys Glu Lys Val Ser Ala 20 25 30 Leu Lys Glu Gly Gly Gly Leu Cys 35 40 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 ammo acids (B) TYPE: ammo acid (C) STRANDEDNESS: Single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Actin 1-28 peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Asp Glu Asp Glu Thr Thr Ala Leu Val Ala Asp Asn Gly Ser Gly Leu 1 5 10 15 Val Lys Ala Gly Phe Ala Gly Ala Asp Ala Pro Arg 20 25 (2) INFORMATION FOR SEQ ID NO: 29. ( ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: ammo acid (C) STRANDEDNESS: smgle (D) TOPOLOGY: unknown (ll) MOLECULE TYPE: peptide (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Anion Exchange Standard peptide (Xl) SEQUENCE DESCRIPTION: SEQ ID NO:29: Glu Tyr Ala Ala Glu Ala Ala Glu Gly Leu Glu 1 5 10 (2) INFORMATION FOR SEQ ID NO: 30: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: unknown (n) MOLECULE TYPE: peptide (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Tnl 104-115 peptide (Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 30: Gly Lys Phe Lys Arg Pro Pro Leu Arg Arg Val Arg 1 5 10

Claims: IT IS CLAIMED 1 A coding segment, for use in an expression vector suitable for expressing heterologous proteins in a host cell, said segment compnsmg (l) a heterologous DNA coding site, (n) a DNA region encoding a first heterodimer-subunit peptide capable of forming an α-hehcal coiled-coil heterodimer with a complementary second heterodimer-subunit peptide. and (in) disposed between the coding site and the DNA region, a cleavage sequence, in frame with said DNA region, encoding an amino acid sequence comprising a target for chemical or enzymatic cleavage, wherein (A) one of said first and second heterodimer-subunit peptides contains at least two heptad amino acid repeat sequences having the form gabcdef. where positions a and d of each amino acid repeat sequence are selected from the group consisting of leucine, isoleucine and valine, and positions e and g of each amino acid repeat sequence are selected from the group consisting of aspartic acid and glutamic acid, (B) the other of said first and second heterodimer-subunit peptides contains at least two heptad am o acid repeat sequences having the form g'a'b'c'd'e'f . where positions a' and d ' of each amino acid repeat sequence are selected from the group consisting of leucine. isoleucine and valine. and positions e and g of each ammo acid repeat sequence are selected from the group consisting of lysine. arginine and histidine. and (C) each of the corresponding d/a ' and a/d ' pairs in complementary heptads of said α-helical coiled-coil heterodimer consists of residues where one of the residues is a valine and the other is selected from the group consisting of leucine and isoleucine 2 The segment of claim 1 , wherein said coding s e includes a multiple cloning site (MCS) at which a heterologous DNA coding region can be inserted 3 The segment of claim 1 , wherein said coding sue includes a heterologous DNA coding region 4 The segment of claim 1 , wherein said DNA region has a sequence selected from the group consisting of the sequences represented by SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 26 and SEQ ID NO 27 5 The segment of claim 1 wherein said cleavage sequence encodes an enterokinase cleavage site 6 A fusion protein, comprising a selected polypeptide having a functional activity, and attached to the N- or C-terminus of the selected polypeptide, a first heterodimer-subunit peptide capable of forming an α-helical coiled-coil heterodimer with a complementary second heterodimer-subunit peptide, wherein (A) one of said first and second heterodimer-subunit peptides contains at least two heptad amino acid repeat sequences having the form gabcdef, where positions a and d of each amino acid repeat sequence are selected from the group consisting of leucine, isoleucine and valine, and positions e and g of each amino acid repeat sequence are selected from the group consisting of aspartic acid and glutamic acid, (B) the other of said first and second heterodimer-subunit peptides contains at least two heptad amino acid repeat sequences having the form g'a'b'c'd'e'f . where positions a' and d ' of each amino acid repeat sequence are selected from the group consisting of leucine, isoleucine and valine, and positions e and g of each amino acid repeal sequence are selected from the group consisting of lysine. arginine and histidine. and (C) each of the corresponding d/a ' and a/d' pairs in complementary heptads of said α-hehcal coiled-coil heterodimer consists of residues where one of the residues is a valine and the other is selected from the group consisting of leucine and isoleucine 7 The fusion protein of claim 6. wherein one of the first and second heterodimer-subunit peptides has a sequence selected from the group consisting of SEQ ID NO 2 and SEQ ID NO 26, and the other peptide has a sequence selected from the group consisting of SEQ ID NO 4 and SEQ ID NO 27 8 A reagent for detecting the presence of an expressed polypeptide, compnsmg a second heterodimer-subunit peptide capable of forming an α-hehcal coiled-coil heterodimer with a complementary first heterodimer-subunit peptide. where the first heterodimer-subunit peptide is expressed in tandem with said polypeptide and a detectable reporter attached to said second heterodimer-subunit peptide, wherein (A) one of said first and second heterodimer-subunit peptides contains at least two 55 heptad ammo acid repeat sequences having the torm gabcdef. where positions a and d of each amino acid repeat sequence are selected from the group consisting of leucine isoleucine and valine, and positions e and g of each amino acid repeat sequence are selected from the group consisting of aspartic acid and glutamic acid, (B) the other of said first and second heterodimer-subunit peptides contains at least two heptad am o acid repeat sequences having the form g'a'b'c'd'e'f , where positions a ' and d' of each amino acid repeat sequence are selected from the group consisting of leucine, isoleucine and valine, and positions e and g of each am o acid repeat sequence are selected from the group consisting of lysine. arginine and histidine, and (C) each of the corresponding d/a' and a/d' pairs in complementary heptads of said α-helical coiled-coil heterodimer consists of residues where one of the residues is a valme and the other is selected from the group consisting of leucine and isoleucine 9 A method of detecting expression of a selected polypeptide, comprising expressing a fusion protein of claims 6 or 7 contacting the fusion protein with a detection reagent composed of a second heterodimer-subunit peptide and a detectable reporter attached thereto, where the first heterodimer-subunit peptide is capable of forming an α-hehcal coiled-coil heterodimer with the second heterodimer-subunit peptide under the conditions of said contacting, and detecting the presence of said heterodimer 10 The method of claim 9, wherein the detection reagent is composed of a fusion protein containing Green Fluorescent Protein in tandem with the second heterodimer- subunit peptide 1 1 The method of claim 9, wherein one of the first and second heterodimer- subunit peptides has a sequence selected from the group consisting of SEQ ID NO 2 and SEQ ID NO 26, and the other peptide has a sequence selected from the group consisting of SEQ ID NO 4 and SEQ ID NO 27 12 A kit for detecting the expression of a selected polypeptide, comprising (I) an expression vector comprising a replication segment which permits replication of the vector in a selected host cell, and an expression cassette which contains, in a 5 -3' direction (a) a promoter functional in said host cell and (b) a coding segment of any of claims 1 -5 where the coding segment may be oriented with either its coding site or DNA region adjacent said promoter, and (II) a detection reagent, comprising said second heterodimer-subunit peptide, where the first heterodimer-subunit peptide is expressed in tandem with said polypeptide, and a detectable reporter attached to said second heterodimer-subunit peptide 13 An affinity matrix composition for the purification of a selected polypeptide, comprising a solid support, and attached to said support, a second heterodimer-subunit peptide capable of forming an α-helical coiled-coil heterodimer with a first heterodimer subunit, where the selected polypeptide is part of a fusion protein of claims 6 or 7 14 The affinity matrix composition of claim 13 wherein the solid support is a glass resm 15 A method of purifying a selected expressed polypeptide comprising (I ) expressing, in a suitable host-cell expression system, a fusion protein containing said selected polypeptide in tandem with a first heterodimer subunit, (n) obtaining from said host cell expression system a suspension containing said fusion protein. (in) passing said suspension over an affinity matrix composition containing (a) a solid support, and (b) attached to said support, a second heterodimer-subunit peptide capable of forming an α-hehcal coiled-coil heterodimer with said first heterodimer subunit, under conditions that promote immobilization of said fusion protein on said matrix composition by way of formation of an α-hehcal coiled-coil heterodimer composed of said first and second heterodimer subunits, (v) washing said affinity matπx while said fusion protein remains bound to said affinity matrix via said α-helical coiled-coil heterodimer, said washing including, in either order, (a) a salt wash step comprising washing said affinity matrix with a solution having a pH of between about 5 5 and about 8 0 and containing between about 0 1 M and about 1 M salt, and (b) an organic wash step comprising washing said affinity matrix with a solution having a pH of between about 5 5 and 8 0 and containing between about 50% and about 100% of an organic solvent, and (vi ) releasing the selected polypeptide from said matrix 16 The method of claim 15, wherein the expression system is an E coli or Yeast expression system 17 The method of claim 15, wherein said releasing includes eluting said fusion protein with a solution having a pH of less than about 3 0 and containing between about 20% and about 80% of an organic solvent 18 The method of claim 15, wherein said releasing includes eluting said fusion protein with a solution having a pH of more than about 10 0 and containing between about 20% and about 80% of an organic solvent 19 The method of claim 15, wherein said organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, tetrahydrofuran, tπfluoroethanol. and acetonitrile 20 The method of claim 19, wherein said organic solvent is acetonitrile 21 The method of claim 15, wherein said salt is selected from the group consisting of sodium chloride, potassium chloride, sodium acetate, ammonium chloride, ammonium acetate, sodium perchlorate, potassium perchlorate, sodium phosphate, and potassium phosphate 22 The method of claim 21 , wherein said salt is NaCl 23 The method of claim 15, wherein one of the first and second heterodimer subunit peptides has a sequence selected from the group consisting of SEQ ID NO 2 and SEQ ID NO 26, and the other peptide has a sequence selected from the group consisting of SEQ ID NO 4 and SEQ ID NO 27 24 A kit for purifying a selected polypeptide, comprising ( I) an expression vector compπsing a replication segment which permits replication of the vector in a selected host cell, and an expression cassette which contains, in a 5'-3' direction (a) a promoter functional in said host cell and (b) a coding segment of any of claims 1-5, where the coding segment may be oriented with either us coding s e or DNA region adjacent said promoter, and (II) affinity matrix composition, containing a solid support, and attached to said support, a second heterodimer-subunit peptide capable of forming an α-hehcal coiled-coil heterodimer with said first heterodimer-subunit peptide, where the selected polypeptide is part of a fusion protein which also contains such first heterodimer subunit 25 The kit of claim 24, further including host cells suitable for use with said expression vector

Inventors: BAUTISTA DAISY, CACHIA PAUL J, HODGES ROBERT S, HOUSTON MICHAEL E JR, IRVIN RANDALL T, TRIPET BRIAN, YU LEI, BAUTISTA, DAISY, CACHIA, PAUL, J., HODGES, ROBERT, S., HOUSTON, MICHAEL, E., JR., IRVIN, RANDALL, T., TRIPET, BRIAN, YU, LEI
Applicants: BAUTISTA DAISY, CACHIA PAUL J, HODGES ROBERT S, HOUSTON MICHAEL E JR, IRVIN RANDALL T, PENCE INC, TRIPET BRIAN, YU LEI, BAUTISTA, DAISY, CACHIA, PAUL, J., HODGES, ROBERT, S., HOUSTON, MICHAEL, E., JR., IRVIN, RANDALL, T., PENCE, TRIPET, BRIAN, YU, LEI
Application year: 1996
Main classification: C12P21
IPC codes: C07K1, C07K7, C07K14, C07K16, C07K19, C12N1, C12N15, C12P21, C12R1