JPH11512620A - Coiled-coil heterodimer methods and compositions for detection and purification of expressed proteins - Google Patents

Abstract

  (57) [Summary] Methods and compositions using complementary heterodimer-subunit peptides capable of forming an alpha-helical coiled-coil heterodimer for affinity purification and detection of the fusion protein.

Description

DETAILED DESCRIPTION OF THE INVENTION               For detection and purification of expressed proteins                Coiled-coil heterodimer method and composition   This application is related to US patent application Ser. No. 08 / 540,397, filed Oct. 6, 1995 Priority, which is incorporated herein by reference). Field of the invention   The present invention relates to a peptide capable of forming an α-helix coiled-coil heterodimer Use for purification and detection of fusion proteins containing one of the paired peptides. About. References   An, G. et al., Mol. Cell. Blol.Two: 1628-1632 (1982).   Ausubel, F.M.CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley an d Sons, Inc., Media PA (1988).   Brasier, A.R., et al., BioTechniques7: 1116-1122 (1989).   Clare, J.J. et al., Bio / Technol.9: 455-460 (1991).   Craxton, M., Methods: A Comparison to Methods Enzymol.Three: 20 (1991).   Cregg, J.M. et al., Mol. Cell Biol.Five: 3376-3385 (1985).   Dykes, C.W. et al., Eur. J. Biochem.174: 411-416 (1988).   Ellis, S.B. et al., Mol. Cell. Biol.Five: 1111-1121 (1985).   Felix, A.M., et al., Int. J. Peptide Protein Res.31: 231-238 (1988).   Gearing, D.P., et al., Bio / Technology7: 1157-1161 (1989).   Germino, J. and Bastia, D., Proc. Natl. Acad. Sci. U.S.A.81: 4692-469 6 (1984).   Gorman, C.M., et al., Mol. Cell. Biol.Two: 1044-1051 (1982).   Gould, S.J. and Subramani, S., Anal. Biochem.7: 5-13 (1988).   Grant, S. et al., Proc. Natl. Acad. Sci.8: 74645 (1990).   Haffey, M.L., et al., DNA6: 565-571 (1987).   Hochuli, E.,GENETIC ENGINEERING , PRINCIPALS AND PRACTICE, VOL. 12(J.S telow) Plenum, NY, pp. 87-98 (1990).   Hodges, R.S., et al., Peptide Res.1: 19-30 (1988).   Hodges, R.S., et al., Peptide Res.Three: 123-137 (1990).   Koutz, P. et al., YeastFive: 167-177 (1989).   Laemmli, U.K. et al. Mol. Biol.80: 575-599 (1973).   LaVallie, E.R., et al. Biol. Chem.268: 23311-23317 (1993).   Lee, K.K., et al., Mol. Microbiol.11: 705-713 (1994).   Maniatis, T. et al.MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Ha rbor Laboratory (1982).   Maxam, A.M. and Gilbert, W., Proc. Natl. Acad. Sci. USA74: 560-564 (19 77).   Miller, J.H.,EXPERIMENTS IN MOLECULAR GENETICS, Cold Spring Harbor La boratory, Cold Spring Harbor, NY (1972).   Mullis, K.B., U.S. Patent No. 4,683,202, issued July 28, 1987.   Mullis, K.B. et al., U.S. Patent No. 4,683,195, issued July 28, 1987.   Nagai, K. and Thogersen, H.C., Nature309: 810-812 (1984).   Nagai, K. and Thogersen, H.C., Methods Enzymol.153: 461-481 (1987).   Porath, J., Protein Exp. and Purif.Three: 263 (1992).   Prasher, D.C. et al., Gene11: 229-233 (1992).   Romanos, M.J., et al., Vaccine9: 901-906 (1991).   Saiki, R.K., et al., Science239: 487-491 (1988).   Sambrook, J. et al.MOLECULAR CLONING: A LABORATORY MANUIAL , 2ND EDITION, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).   Sarin et al., Anal. Biochem.117: 147-157 (1981).   Skerra, A. et al., Biotechnology9: 273 (1991).   Towbin, M. et al., Proc. Natl. Acad. Sci. USA76: 4350 (1979).   Yanisch-Perron, C. et al., Gene33: 103-119 (1985). Background of the Invention   One of the major commercial applications of biotechnology is the recombinant expression of proteins. Epo Etin α, G-CSF, somatotropin, insulin, tPA, urokina And pro-urokinase, various interferons, EPO, and specialty enzymes Several recombinant proteins, including specialty enzymes, are well established Has a commercial market (bioprocess engineering).   There are three general technical hurdles to recombinantly produce a protein I do. The coding sequence of the protein must be available and the gene Must be cloned into an expression vector; Suitable host cells and media for expressing and, preferably, secreting the protein. Nutrient conditions must be found; and the expressed protein must be Isolated to produce a final product (eg, a product suitable for pharmaceutical use), and In some cases it must be treated enzymatically.   The purpose was to find favorable conditions for high levels of protein expression and secretion. For a given expression system, the expression, level of expression, and expressed protein It would be desirable to be able to monitor quality localization. Ideally, such a monitor Can be performed without modifying the expressed protein, and Ju forms, and various in vitro forms (eg, dot blot or waste cloth) Tan blot analysis).   For the purpose of isolating the expressed protein, the protein is It is desirable to produce the protein in a form that is suitable for affinity purification. Previously proposed approaches for this purpose have reduced proteins to small It is expressed in tandem with the peptide antigen segment. Then, the expressed tag The protein to its specificity on a support matrix with antibodies specific for the antigen Purification based on specific binding. For large-scale production, this approach is Requires an amount of antibody that is proportional to the amount of protein produced, Costs can be greatly increased. To release bound proteins from a solid support The need for antigen competition can also increase the cost of the purification process. Summary of the Invention   The present invention relates to a replication seg that enables the replication of a vector in a selected host cell. And an expression cassette comprising an expression cassette. The cassette is Promoter and coding segment functional in host cell in 5'-3 'direction including. This coding segment is then replaced by the heterologous DNA coding site, and the complementary Second heterodimer-subunit peptide and α-helical coiled coil A first heterodimer-subunit peptide capable of forming a heterodimer DNA region to be loaded. In addition, this coding segment contains the coding region and the DNA region. Region, a cleavage sequence that is in-frame with the DNA region, Alternatively, a cleavage site encoding an amino acid sequence that provides a target for enzymatic cleavage May be included. This coding segment is located next to the coding site or promoter. It can be oriented with any of the flanking DNA regions.   In a preferred embodiment, the (first and second heterodimer-subunits) One of at least two heptadiamides having the form of gabcdef A non-acidic repeat sequence, wherein positions a and d of each amino acid repeat are leucine, Selected from the group consisting of isoleucine and valine, and Row positions e and g are selected from the group consisting of aspartic acid and glutamic acid. It is. The other (of the first and second heterodimer-subunit peptides) is g It contains at least two heptad amino acid repeats in the form of 'a'b'c'd'e'f'. Where the positions a ′ and d ′ of each amino acid repeat are leucine, isoleucine, And valine, and positions e and And g are selected from the group consisting of lysine, arginine, and histidine. This In a preferred embodiment, the corresponding d / a ′ and a / d ′ in the complementary heptad In each of the pairs, one of the residues is valine and the other residue is leucine and It consists of residues selected from the group consisting of soleucine.   The cloning site is preferably a multiple cloning site (MCS) (where different (A species DNA coding region may be inserted). Such heterologous DNA coding regions are Typically, it encodes a selected polypeptide of interest. Selected Polypeptide The DNA encoding the code is typically inserted at the cloning site, resulting in Is the DNA region encoding the first heterodimer-subunit peptide (this (The sequences represented by SEQ ID NO: 2 and SEQ ID NO: 4). Expressed in frame.   In a related aspect, the invention relates to expression of a heterologous protein in a host cell. Codesets for use in suitable expression vectors (eg, the vectors described above) Including mentament. The segments are heterologous DNA coding sites and complementary as described above Heterodimer-Subunit Peptide and α-Helix Coiled Coil DNA encoding a heterodimer-subunit peptide capable of forming rhodimers Including the region. In addition, this segment, between the coding site and the DNA region, A cleavage sequence encoding an amino acid sequence containing a target for selective or enzymatic cleavage, It can be included in frame with the DNA region. The coding site is either 5 'or 3' of the DNA region It can be located in somewhere. Cloning sites and coding sequences are Including the embodiments described above.   Coded by the above code segment or such code segment Produced by an expression vector containing the fragment (for example, the expression vector described above). Also included in the invention are fusion proteins. Fusion proteins have functional activity Polypeptide (ie, the selected polypeptide of interest), and the pepti Heterodimer-subunit pairs of the above type attached to the N-terminus or C-terminus of the The peptide (ie, the E coil, 0993, K coil, or 0994 peptide).   Reagents for detecting the presence of the expressed fusion protein are also included in the invention. You. Reagents are used for the heterodimer-subunit peptide ( Α-helix coiled-coil heterodimer with the first coil peptide) Obtained second heterodimer-subunit peptide (second coil peptide) It is composed of The reagent was a fusion protein containing a heterodimer-subunit peptide. Expression of the protein (eg, as described above) may be determined by the Heterodimer or heterodimer between the immer-subunit peptide and the detection reagent Forming a rhodimer complex and detecting the presence of heterodimer formation Useful for detecting by   A kit for detecting the expression of a selected polypeptide is a heterodimer- Code seg described above for expressing fusion proteins containing subunit peptides And the detection reagent described above.   In yet another aspect, the invention provides an method for purifying a selected polypeptide. An affinity matrix composition. The composition comprises a solid support and this support. Heterodimer-subunit peptides of the type described above (coil peptide (E.g., SEQ ID NO: 4 or SEQ ID NO: 27). The composition is described below As described in the method for purifying the selected expressed polypeptide. It is for.   The invention also includes a method of purifying the selected expressed polypeptide. This one The method comprises the steps of (i) running in a suitable host cell expression system (eg, an E. coli or yeast expression system). The selected polypeptide is then converted to a first heterodimer subunit (eg, Expressing the fusion protein comprising tandem with SEQ ID NO: 2 or SEQ ID NO: 26), (Ii) obtaining a suspension containing the fusion protein from a host cell expression system; And (iii) passing the suspension through the affinity matrix composition described above. Including the process. Next, the affinity matrix is used to determine whether the fusion protein is α-helical. Binding to the affinity matrix via a coiled-coil heterodimer Washed as is. This washing step can be performed in any order, with (a) about 5.5 and about 8. A pH between about 0.1 M and about 1 M (eg, sodium chloride). , Potassium chloride, sodium acetate, ammonium chloride, ammonium acetate, Sodium chlorate, potassium perchlorate, sodium phosphate, and potassium phosphate Washing the affinity matrix with a solution containing A salt washing step, and (b) having a pH between about 5.5 and 8.0, and about 50% and about 100 % Organic solvents (eg, methanol, ethanol, isopropanol, (Trahydrofuran, trifluoroethanol, and acetonitrile) Organic washing step including a step of washing the affinity matrix with a solution including. After washing, the selected polypeptide is either eluted or enzymatically cleaved. Depending on the release from the matrix. Preferably, the elution solution is about 3.0 or Has a pH lower or about 10.0 or higher. Exemplary elution The solution contains between about 20% and about 80% of the organic solvent.   Kits for obtaining the protein expressed in purified form are available in the types described above. For expressing a fusion protein having a telodimer-subunit peptide Expression systems described above and fusion proteins expressed to solid supports in compositions Affinity matrix composition of the above type for affinity binding of including.   These and other objects and features of the invention are set forth in the following detailed description of the invention. It is more fully understood when read in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE FIGURES   FIGS. 1A, 1B, 1C, 1D, 1E, and 1F show affinity purification by the method of the present invention. Is shown below.   Figure 2 shows peptide 0993 in the α-helix heterodimer configuration (SEQ ID NO: No. 26) and 0994 (SEQ ID NO: 27) are shown as helical wheel representations .   3A, 3B, 3C, 3D, and 3E show two heterodimers in a parallel configuration -Schematic representation of adjacent heptads of subunit peptides, showing homodimer vs. Stabilizing / destabilizing effects of charged residues at positions e and g in telodimers Are compared. FIG.3A shows oppositely charged residues at positions e and g of the heptad. Thus, a stabilized homodimer is shown. FIG.3B shows the position of the heptad at positions e and g. 2 shows a heterodimer destabilized by oppositely charged residues. FIG. Homoda destabilized by positively charged residues at positions e and g of the heptad Indicates an immer. FIG.3D shows similar charged residues at positions e and g of the heptad. Shows a stabilized heterodimer. FIG.3E shows the position of the heptad at positions e and g. 1 shows a homodimer destabilized by negatively charged residues.   Figures 4A, 4B, and 4C are designed to form coiled-coil heterodimers Has a positive or negative charge at positions e and g in the peptide FIG. 3 shows a schematic diagram of some possible distributions of heptad. FIG. 4A shows alternating positive and negative From Heterodimer-Subunit Peptides with Consecutive Charged Heptads 1 shows a schematic diagram of the heterodimer thus constituted. Figure 4B shows the heterodimer-subunit Peptide (one of which has a predominantly positively charged heptad, and The other has a predominantly negatively charged heptad) FIG. FIG. 4C shows the heterodimer-subunit peptide (one of them). One has an all positively charged heptad and the other an all negatively charged heptad FIG. 1 shows a schematic diagram of a heterodimer composed of   5A, 5B, 5C, 5D, 5E, 5F, and 5G show heterodimer-subunit peptides. 1 shows a schematic diagram of the synthesis of a DNA fragment encoding a tide. FIG.5H shows plasmid p 3 shows a map of RLD-E.   FIG. 6A shows the DNA fragment inserted at the multiple cloning site (MCS). N-terminal heterodimer-subunit of selected proteins encoded by Expression vector expression cassette useful for expressing a fusion protein having a peptide To indicate   FIG. 6B shows the DNA fragment inserted at the multiple cloning site (MCS). Heterodimer-subunit at the C-terminus of selected proteins encoded by Expression vector expression cassette useful for expressing a fusion protein having a peptide To indicate   FIG. 7A shows a reverse phase (RP) -HPLC chromatogram of peptide 0993 (SEQ ID NO: 26). You.   FIG.7B shows the RP of the flow-through fraction of the selective dimerization affinity column of the present invention. -Shows the HPLC chromatogram.   FIG. 8 shows a 60% acetonitrile wash of the affinity column described in FIG. 7B. 2 shows an RP-HPLC chromatogram of the present invention.   FIG. 9 shows RP-H of the next GndHCl eluted fraction of the affinity column described in FIG. 3 shows a PLC chromatogram.   FIG. 10 is used to test the specificity of the affinity matrix of the present invention. Three test charged peptides (SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30) 2 shows a chromatogram of a mixture of   FIG. 11 shows an affinity column loaded with the test peptide shown in FIG. 2 shows the chromatogram of the 0.2 M KCl washed fraction of FIG.   Figure 12 shows a 0.5 M KCl wash of the affinity column loaded with the test peptide. 2 shows the chromatogram of the minutes.   FIG. 13 shows the chromatogram of the 1.0 M KCl washed fraction of the affinity column shown in FIG. Show ram.   FIG. 14 shows a chromatogram of the next 1.0 M KCl wash of the column described in FIG.   FIG. 15 shows the next 5.0 M GndHCl / 50 mM K following the column described in FIG._TwoPO_FourIs shown.   FIG. 16A shows PAK (128-144) / Ecoil fusion protein stained with Coomassie Blue. 1 shows a quality SDS-PAGE gel.   FIG. 16B shows a plate with either the coil K reporter or the monoclonal antibody PK99H. Western blot analysis of the lobed PAK (128-114) / Ecoil fusion protein 2 shows ligand and ligand blots.   FIG. 17 shows the load sample and the fraction eluted from the affinity column of the present invention. Shows a series of RP-HPLC chromatograms, where the load fraction was recombinantly expressed. Was a crude periplasmic extract containing the E-coil peptide.   FIG. 18 shows a recombinant from crude on an affinity column made according to the invention. A series of RP-HPLC chromatograms of the purified Pak-cili-E coil peptide expressed Show.   FIG. 19 shows an ELISA assay using a reporter molecule formed according to the present invention. Show. Detailed description of the invention I.Definition   Unless otherwise indicated, the following terms have the following meanings.   The term “peptide” refers to a polyamide chain based on amino acids. This chain is It can vary in length from amino acids to about 100 amino acids.   The term “polypeptide” refers to a polypeptide based on amino acids that includes at least two amino acids. Represents an amide chain.   "Promoter functional in host" refers to the vector in the selected host DNA in an expression vector, which results in enhanced transcription of the adjacent 3 'downstream coding sequence in E. coli Means an array.   The "coding segment" in the expression vector is a DNA coding for the polypeptide. Refers to a segment.   "Heterologous DNA coding site" refers to (i) one or more restriction sites in the expression vector. (Eg, a multiple cloning site) in which the selected heterologous protein A site into which DNA encoding the protein can be inserted, or (ii) the encoding DNA itself Say. "Heterodimer-subunit peptide" One of two complementary peptide subunits capable of forming an yl heterodimer Say. Further, the term “heterodimer polypeptide” or “heterodimer "Polypeptide complex" refers to two linked, non-identical polypeptide chains.   Unless otherwise indicated, the sequences of peptides and polypeptides are Given in order to the ruboxi end. All amino acids identified herein Residues are natural, ie, L-shaped, unless otherwise specified. Of standard peptide Consistent with the nomenclature, the abbreviations for amino acid residues use standard, commonly used in the art. A three-letter and / or one-letter code.   In the context of a "derivatized" polypeptide subunit, the term "derivatization" One or more covalently linked to one or more amino acid residues forming a subunit Polypeptide subunit having a functional or reporter portion of Understood. Here, this part is used before (i) synthesis of the polypeptide subunit. Or later, linked to one or more amino acid residues in the subunit Or (ii) extending the peptide subunit, for example, at the N-terminus of the subunit. May form a length. In addition, the functional or reporter moiety may Subunit directly or with a linker or spacer (eg, polyglycyl Spacers).   The term “benign medium”, as used herein, is typically about 6 and about 8 Physiologically compatible, with a pH between and a salt concentration between about 50 mM and about 500 mM Aqueous aqueous solutions are described. Preferably, the salt concentration is between about 100 mM and about 200 mM . An exemplary benign medium, called Buffer A, has the following composition: 50 mM potassium phosphate Um, 100 mM KCl, pH 7. An equally effective benign medium is, for example, sodium phosphate By replacing potassium phosphate with and / or KCl with NaCl. obtain. II.General appearance of heterodimer-subunit peptides   The complementary heterodimer-subunit peptide has two non-identical peptides. Chain, typically about 21 to about 70 residues in length, and a double-stranded alpha helical Have amino acid sequences compatible with their formation into heterodimeric coiled coils doing. These are generally referred to herein as HSP1 (heterodimer-subunits). Knit peptide 1) and HSP2 (heterodimer-subunit peptide 2) It is expressed as Since peptides can form α-helical coils, they also In this specification, it is also called "coil peptide".   In a benign aqueous medium, the isolated heterodimer-subunit peptide Typically, it is a random coil. HSP1 and HSP2 are replaced by α-helix coiled coils When they are mixed together under conditions that favor the formation of Using a double-stranded α-helix coiled-coil heterodimer complex (HSP1-HSP2 ).   The peptides in the α-helical coiled-coil conformation Interact with each other. This mode is determined by the primary sequence of each peptide. It is. The tertiary structure of the α-helix consists of seven amino acid residues in the primary sequence. It seems to correspond to about two revolutions of the box. Therefore, α helix conformation The primary amino acid sequences that give rise to amino acids are each in units of 7 amino acid residues (heptad and Call). Heterodimer-subunit peptides are a series of tandem It consists of a simple heptad. Heptad sequence is a specific heterodimer-subunit When repeated in a knit peptide, this heptad can be referred to as a “heptad repeat”. Or simply "repeat".   Identification at defined locations in each heptad as detailed below Is a double-stranded α-helix coiled-coil heterodimer Acts to stabilize the structure or complex.   HSP1 and HSP2 also bind to the α-helix or coiled-coil Reaction to stabilize properties (either within or between helices) May be included. III.Characteristics of heterodimer-subunit peptide   Complementary heterodimer-subunit peptides (HSP1 and HSP2) are representative Typically, they are similar in size, each generally being about 21 to about 70 residues long (3 to 10 heptads). Tado) range.   Peptides can be prepared by various methods known to those of skill in the art, including chemical and recombinant methods. ). For example, the ABI model 430A peptide synthesizer was previously s et al. (1988) and as described in Example 1 in conventional t-Boc chemistry. Can be used. Alternatively, the peptides can be used, for example, as detailed in Examples 5 and 6. As described above, expression in an appropriate host cell system using an expression vector encoding the peptide. Can be revealed.   Subsequent to synthesis, the peptide may be prepared by any of a number of methods known to those of skill in the art, for example, As detailed in Example 1, reverse phase high performance liquid chromatography (RPC) and It can be purified using a "SYNCHROPAK" RP-P column.   The composition and purity of the peptide can be determined by several methods, as detailed in Example 1. (Amino acid composition mass spectrometry using a Beckman model 6300 amino acid analyzer and “BIOION- 20 ”including molecular weight analysis using time-of-flight mass spectrometry at Nordic) obtain.   A.Coiled-coil heterodimer formation   HSP1 and HSP2 dimerization occurs in the primary amino acid sequence of each peptide. Caused by the presence of repeated heptad motifs of conserved amino acid residues. Each The individual positions in these heptads are, for HSP1, as shown in FIG. Represented by the letters ag and for HSP2 by a'-g '. HSP2 location ( For example, a ', g') are the following heterodimer-subunit peptides or In the general discussion of heptad positions in coiled peptides, the (') Mentioned without issue.   Repeated heptad motifs with the appropriate amino acid sequence are represented in Part D below. Under acceptable conditions, the HSP1 and HSP2 polypeptides are converted to the heterodimeric α Guided to be assembled into a helix coiled coil structure. Individual α helicopter The peptides are defined as positions a and d of the respective heptad, where They contact each other along their respective hydrophobic surfaces.   HSP1 and HSP2 are heterodimeric in either parallel or antiparallel configurations. -Assembled into a coiled coil helix (coiled coil heterodimer) Can be In a parallel configuration, the two heterodimer-subunit peptides Ricks, they have the same orientation (from amino terminus to carboxyl terminus) Are arranged as follows. In an antiparallel configuration, the helix is one helix The amino terminus is aligned with the carboxyl terminus of the other helix, and The converse is also arranged to be the same.   A diagram of the relative directions of the positions ag of the two interacting α-helices is shown in FIG. Show. This figure shows two exemplary heterodimers arranged in a parallel configuration- Subunit peptides, 0993 (E; SEQ ID NO: 26) and 0994 (K; SEQ ID NO: 27) , A true direction schematic of the first two revolutions (one heptad) of FIG.   Heterodimer-subunits designed in accordance with the guidance provided herein The knit peptide is typically assembled into an assembly parallel to the anti-parallel direction. Indicates a slight preference. However, in general, two heterodimer-subunit pairs The direction in which the peptide forms the α-helical coiled coil (parallel versus antiparallel) Rhodimer-their ability to support together the moieties bound to the subunit peptide Is not particularly relevant.   In FIG. 2, amino acids are circled and indicated by the one-letter code. So Thus, consecutive amino acid positions are numbered and from N-terminal to C-terminal They are connected by a straight line with an arrow indicating the direction. The interaction between the two helices is Indicated by arrows. The wide arrow between the helices indicates the position a of the adjacent helix. 9 shows a hydrophobic interaction with position d. E and g positions of adjacent helices Interactions between the helix are indicated by curved arrows above and below the helix bond Is done.   B.Hydrophobic interactions in coiled-coil heterodimer stability   The hydrophobic interaction between the helices is caused by the heterodimer-subunit peptide Due to the hydrophobic residues at positions a and d. Residues at these positions link the helix It is effective in maintaining contact with leucine, isoleucine, valine, Enylalanine, methionine, tryptophan, tyrosine, alanine, and And derivatives of any of the foregoing. Alanine, cysteine, serine, threonine, Other residues including asparagine and glutamine are also found in some heptads. May occupy position a or d as long as the others are occupied by hydrophobic residues.   Proper selection of particular residues to occupy positions a and d is an important part of the invention. Plane. If the hydrophobic interaction is too strong, then similarly charged residues will be in the e and g positions. A significant proportion of helices, even when present in Are formed as homodimers at pH 7 (see Part C below). on the other hand , The residues at positions a and d are selected such that the hydrophobic interaction is too weak ( For example, Ala at both positions, the helix forms a coiled-coil dimer at all I can't. Preferably, the residue pair at positions a and d is ≧ 95% More preferably ≧ 99% selected to promote heterodimer formation You. The degree of heterodimer formation relative to homodimer formation was determined, for example, in Example 3. Can be measured as described in   Experimental results performed in support of the present invention show that all heptads of both coil peptides All positions a and d of the tado are due to Ile, Leu, or any combination thereof. Stabilizes the heterodimer, even if detailed herein Even if the e- and g-positions designed to contain charged residues The effect is strong enough to stabilize homodimers and heterodimers. Indicates that there is. In addition, such homodimers have only three heptads. Formed between the containing coil peptides. According to an important aspect of the present invention, such a Homodimer formation is minimized or eliminated for practical purposes It should be. The presence of a substantial proportion of homodimers makes it difficult to practice the invention Or impossible. Because the coil peptide ( That is, a suspension containing a fusion of the selected protein and the coil peptide Used to derivatize coiled peptides in, or solid matrices Coil peptide in suspension to be used), the component solutions are used for their intended purpose. Because it assembles itself before it can be done.   Several strategies have been developed for the hydrophobic interaction between residues at positions a and d. Strength, interaction is important to maintain heterodimers throughout the protein purification procedure Strong enough, but they are not strong enough to stabilize homodimers Can be used to adjust or set up. The preferred strategy is all Is a coil peptide with Leu at position d and Val at position a, or Or all heptads have Ile at position d and Val at position a. The use of tide. Another strategy is to have all heptads in position a Leu and a coiled peptide with Val at position d or all heptads By using a coil peptide having Ile at position a and Val at position d. is there. Modifications of these strategies are described in the exemplary heterodimer-subunit Peptide E coil (SEQ ID NO: 2) and K coil (SEQ ID NO: 4), and peptides Used in the design of Tide 0993 (SEQ ID NO: 26) and 0994 (SEQ ID NO: 27). Note that Another strategy is to use all positions a and d, one of which is occupied by Ile and Leu, and Use destabilizing residues (eg Ala or Asn) for remaining positions a and d It is. This remaining position is preferably for each coil rather than either end Close to the center of the peptide.   C.Ionic interactions in coiled-coil heterodimer stability   The α-helix dimer coiled coil conformation is shown in FIG. Ionic phase between the residues at position e and g of the adjacent helix, as It can be stabilized by interaction. The other elements are equivalent and each of the dimers The helix loads positively charged residues at one position (eg, e) and negatively When having charged residues at other positions (eg, g), homodimer formation is advantageous. Yes (FIG. 3A: in comparison to the heterodimer in FIG. 3B). But each herrick Form homodimers if they have similarly charged residues at both positions ( Instead of Figs. 3C and 3E), two oppositely charged helices combine to form a heterodimer. (Figure 3D).   The conformation of polypeptides (eg, HSP1 and HSP2) in solution is It can be determined from the CD spectrum of the solution. These data are for individual peptides Information about your conformation (random coil versus α helix), For example, a heterodimer complex to a homodimer complex of HSP1 and HSP2 Provides information about the relative mass of the body. Example 2 is one method of measuring a CD spectrum. The method will be described in detail. Example 3 describes how to use CD spectroscopy to measure Whether the conformation of the tide can be evaluated will be described in detail.   In the diagram shown in FIG. 2, the ionic interaction between the two helices is HSP1 (0993; arrangement). Negatively charged (Glu) residues at positions e and g of SEQ ID NO: 26) and HSP2 (0994; sequence No. 27) and positively charged (Lys) residues at positions e and g.   The negatively charged residue may be aspartic acid, glutamic acid, or a derivative thereof. Can be Positively charged residues can be lysine, arginine, histidine, or These derivatives may be used.   D.Advantageous conditions for coiled coil formation   Guidan composed of repeating heptads and represented in Parts A-C above Heterodimer-subunit peptides designed according to the procedures described in Part I above Easily forms a coiled-coil heterodimer in a benign medium as defined in The degree of α-helix coiled-coil heterodimer formation is, for example, as described in Example 3. Can be determined from the CD spectrum, as described above.   Coiled-coil heterodimers are available at pH and salt ranges given for benign media. Some molecular phases of heterodimers versus homodimers may form under external conditions Interactions and relative stability may differ from the properties detailed above. For example, Ionic interactions between the e and g positions that tend to stabilize heterodimers Can be, for example, protonation of the Glu side chain at acidic pH, or L at, for example, basic pH. Deprotonation of the ys side chain can be destroyed at low or high pH values.   However, the above low and high pH values for coiled-coil heterodimer formation The effect of the value can be overcome by increasing the salt concentration. Increase salt concentration Can neutralize or destabilize stabilizing ionic interactions Ionic repulsion can be suppressed. Certain salts may neutralize ionic interactions Has greater efficacy. For example, ClO with a concentration of 1M or more_Four-Anion Is the largest α-helix structure (CD measurement performed as detailed in Example 2). May be sufficient to induce In order to achieve this, Cl^-Ions may be required. At low and high pH The effect of high salt on ildocoil formation is also due to the helical ionic attraction between helices. It is not essential for the formation of the coil, but rather the coiled coil is formed as a heterodimer. Is a factor in the tendency to be formed as a homodimer Is shown.   E. FIG.Heptad changes in heterodimer-subunit peptides   Part A, Part B and Part C above are the heterodimer-subunit peptides. At a particular position in the heptad of a peptide, any amino acid residue may be included, but not Guidelines for which amino acid residues are preferred. This peptide Typically forms an α-helical coiled-coil structure in a benign medium Resulting in the peptide. This part is based on the guidelines shown in Part A to Part C above. Heptads having a sequence according to how the heterodimer-subunit Some examples of how they can be placed within the peptide are described.   The heterodimer-subunit peptide of the present invention has three or more Of heptads. The sequences of each of these heptads are all identical. Or they may be different. In addition, the sequence of the internal repeat Depends on whether the repeat contains an amino acid coupling residue (eg, cysteine). Can differ from each other. Such coupling residues are, for example, heterodimers Mer-subunit peptide to resin support matrix or another polypeptide Can be used to tether.   Two Heterodimers-Sub of α-helical Coiled Coil Heterodimer Pair Significant interactions between unit peptides are adjacent in each peptide The heterodimer-subunit peptide exists between the "complementary" heptads. The primary sequence of the heptad in the peptide is such that the residue in each heptad is the second heterodyne. Advantageously interacts with residues in the complementary heptad of the immer-subunit peptide It can change as long as you use it.   The adjacent heptad is then, for example, a heterodimer-subunit peptide. The net charge on the polypeptide is the α-helix heterodimer coiled coil In the sequence so that it can be modified without affecting the ability to form You. This relationship is shown in FIG. This figure shows three examples of CP dimer pairs. Each Each heterodimer-subunit peptide has five heptads. It A + or-sign in each heptad indicates two charges (one Is in position e and one is in position g). Adjacent complementary heptads carry opposite charges Note that it has. For the purposes of this example, each heptad Positions other than the e and g positions are assumed to have a total net charge of zero. In FIG. 4A HSP1 and HSP2, which form a dimer in The excess of peptad and one negatively charged heptad resulted in +2 and It can be recognized that it has a net charge of -2. Similarly, in FIG. 4B, HSP1 and HSP2 has a net charge of +6 and -6, respectively, and HSP1 and FIG. And HSP2 have a net charge of +10 and -10, respectively. About this scheme All other modifications are, of course, possible without departing from the spirit of the invention.   G. FIG.Heterodimer-a moiety that couples to a subunit peptide   Coupling residues (eg, amino acid coupling residues) can be Can be incorporated into one and both coiled peptides to facilitate use of the peptide. You. Coupling residues are located in the central region of the peptide and / or at one or both ends Can be taken into account. If a coupling residue is present in the central region, this residue The group is typically located at the b, c, and / or f positions of one or more heptads. Preferably, it is taken into the f-th position. These positions are used for coiled-coil heterodimers. Located along the outer surface of the The coupling residue at one or both ends is typically Couples to the terminal residue.   Preferred coupling groups are thiol groups of cysteine residues. This is It can be easily modified by standard methods. Example 4 presents in the coil peptide How to use the cysteine thiol residue in other peptides at these positions It will be described in detail how to combine.   Other useful coupling groups are the thioester group of methionine and the histidine group. Midazole groups, arginine guanidyl groups, tyrosine phenol groups, and Contains the indolyl group of liptophan. These coupling groups are known to those skilled in the art. Can be derivatized in a similar manner as detailed in Example 4 using the reaction conditions of .   H.Exemplary coil peptide   E coil (SEQ ID NO: 2) and K coil (SEQ ID NO: 4) heterodimer-sub Unit peptides and 0993 (SEQ ID NO: 26) and 0994 (SEQ ID NO: 27) are examples Exemplary HSP1 and HSP2 heterodimer-subunit peptides. Both pairs The peptides contain a Val residue at their position a and a Leu residue at their position d. Is effective for stabilizing coiled-coil heterodimers, but homologous Hydrophobic interactions that are not strong enough to overcome electrostatic repulsion between dimers to be certain.   The e- and g-positions of the 0993 heptad contain a Glu residue, while the e-position of the 0994 heptad And the g-position contains a Lys residue. The corresponding in the complementary heptad of 0993 and 0994 The charge opposite the position is described above and is illustrated in FIGS. 3A-E and FIG. Thus, the α-helical coiled-coil heterodimer is stabilized.   In a similar manner, the charged groups at positions e and g prevent the formation of homodimers and And destabilize it. According to one aspect of the invention, this destabilization is To overcome the hydrophobic interaction that exists between properly selected residues at a and position d Strong enough to help form both heterodimers and homodimers. You.   As described above, two α-helical coiled-coil heterodimer pairs The prominent interaction between the heterodimer-subunit peptide is significant for each peptide. Between adjacent complementary heptads of the tide. In a preferred embodiment of the invention , Each corresponding d / a ′ and a / d ′ pair in such a complementary heptad is One of the residues is valine and the other is from leucine and isoleucine. Consisting of residues selected from the group consisting of This relationship can be seen in FIG. This Here, the d / a ′ pair consists of Leu (position d) and Val (a ′ position), and the a / d ′ pair is Val (Position a) and Leu (position d). IV.Using heterodimer-subunit peptides for purification of fusion proteins Protein expression vector   The present invention relates to a coil peptide and a selected polypeptide expressed in tandem. Expression of fusion proteins (ie, heterodimer-subunit peptides) And expression vectors useful for Then, the coil peptide portion of the expression fusion Purifies proteins using an affinity matrix (as described below) Or in situ (eg, fluorescence detection in cells) or tan Protein detection systems (eg, enzyme-linked immunosorbent assay (ELISA) and / or Or dot blot, slot blot, or western blot) It can be used to detect proteins. The fusion protein is a detection reagent (This may be, for example, a reporter-labeled coil that is complementary to the coil peptide in the fusion. (Including peptides).   How to clone a selected sequence into an expression plasmid or vector (Eg, Maniatis et al., 1982; Ausubel et al., 1988). Next Such plasmids or vectors are then used in suitable host cells (eg, bacteria or Or yeast) and cells are induced to produce the recombinant polypeptide. Produce. Depending on the expression system, the medium from which the cells are derived or the cells themselves may be recombinant This polypeptide is used to make a suspension containing the polypeptide, and then Tides can be purified using the methods of the present invention.   For illustration, Example 5 describes two sets of complementary overlapping oligonucleotides. A recombinant construct of the E coil gene using nucleotides is described in detail. Each session Anneal the oligonucleotides in the digest and digest with the appropriate restriction enzymes. To generate a sticky end at one end of each double-stranded fragment (ds) (FIG. 5C and 5F) and ligate the two ds fragments using T4 DNA ligase to 177 bases A pair of single ds fragments was generated (FIG. 5G). The 177 bp fragment was purified , And digested with EcoRI and BamHI for cloning into plasmid vectors The appropriate 166 bp fragment was generated.   The construction of an exemplary plasmid expression vector pRLD is described in Example 6. Expression The polylinker site of vector pASK40 (Skerra et al., 1991) was modified with pHIL-S1 and PIC9 (I nvitrogen, San Diego, CA) Thus, pRLD was constructed. This modification can be digested to shift the reading frame. And clone the pASK40 cloning site more than that of the Pichia pastoris vector. Designed to match. Next, the sequence encoding the E coil gene was replaced with pRLD Into the E. coli E coil vector pRLD-E (FIG. 5H).   Expression vectors allow autonomous replication of the vector in a selected host cell It may include a replication segment and an expression cassette. The cassette is in the 5'-3 'direction, Contains a promoter and coding segment functional in the host cell, , Described in detail below. Vectors can also contain additional elements (eg, Sequence encoding a selectable marker that ensures the maintenance of the vector in the cell) Properly located ribosome binding site downstream of the motor, and the 3 'end of the cassette It may include an end transcription termination (TT) sequence.   Elements suitably included in the expression vector are specific to the vector used. Depends on the expression system. For example, an element typically present in an E. coli expression vector Encodes a selectable marker that ensures (i) the maintenance of the vector in the cell Sequence (eg, ampicillin resistance gene), (ii) Controllable transcription factor that can produce large amounts of mRNA from the selected gene (Iii) a translation control sequence (eg, an appropriately located ribosome binding site) Position and initiator ATG), and (iv) selection in the correct orientation in the vector. Polylinker or multiple cloning site to simplify insertion of inserted genes (MCS).   Examples of inducible promoters suitable for use in E. coli expression vectors include inducible promoters. Can produce large amounts of mRNA from a gene operably linked to a promoter. And Iac, trp, and tac. Suitable for use in E.coli expression vectors Other promoters are the highly efficient phage T7 gene 10 promoter (this is , Using T7 RNA polymerase) and the powerful bacteriophage pL promoter (Ausubel et al., 1988).   The recombinant polypeptide or fusion protein is typically a recombinant polypeptide. The appropriate E. coli in the expression plasmid encoding the coli expression strain ( For example, it is expressed by transforming JM83). Examples of suitable transformation methods Heat shock treatment (Yanisch-Perron et al., 1985) and electroporation Option. The transformed cells are then typically plated on selective agar plates. Plate (eg, an LB plate supplemented with carbenicillin (100 μg / ml)). Cells containing the appropriate recombinant DNA are subjected to restriction digest analysis and subsequent Confirmed by DNA sequencing of the miniprep plasmid DNA.   E. coli with the appropriate DNA selected E. coli cloned cells are typically Density (for example, 0.5 A₅₅₀(Eg, at 25 ° C, with shaking) LB medium containing 100 μg / μl carbenicillin). Induction (eg with IPTG If using a plasmid that requires a Derived from   Suspensions containing the expressed protein can be prepared by methods known in the art (eg, Modified osmotic shock treatment (Ausubel et al., 1988)). E. in coli Proteins that are expressed in large quantities in are sometimes precipitated in insoluble aggregates (inclusion bodies), Such proteins can be prepared, for example, using solubilization in denaturing agents known to those of skill in the art. Can be recovered (Ausubel et al., 1988).   Other types of host cell expression systems can be used in the practice of the present invention. For example, yeast (eg, Pichia pastoris) has very high expression levels, It offers a number of advantages, including ease and some post-translational modifications. Yeast expression vector Vector can be used to transform yeast spheroplasts, and The transformed cells can be used to produce a recombinant polypeptide. Yeast expression vector Exemplary inducible promoters suitable for use in vector And GAL1, GAL4, GAL7 and GAL10 promoters. Example 7 Describes the use of yeast expression vectors in the practice of the present invention. E coil gene The containing DNA fragment was PCR amplified and plasmid pIC9 (Invitrogen, San Di ego, CA) to generate the Pichia E coil vector p9CE. Sure Transform the identified yeast plasmid into Pichia pastoris and select yeast cells. Selected, induced and treated as described in Example 7 to contain the recombinant protein. The resulting culture supernatant was generated.   The expression cassette or coding segment of the present invention may also be used for baculovirus systems. Can be used to express the fusion protein. Where is expressed Genes for proteins to be replaced with insects instead of highly expressed non-essential genes Inserted into the virus. The foreign protein is a recombinant virus in cultured insect cells. (Ausubel et al., 1988). Vector like this The vector typically integrates into the host cell genome via homologous recombination Recombination sequences that allow the Used in these vectors The specific promoter is generally the identity of the non-essential gene that is excised (iden tity). Exemplary promoters include the polyhedrin promoter (Usually used to drive expression of the AcMNPV polyhedrin gene) and And p10 promoter (Ausubel et al., 1988).   Mammalian cells also use the expression cassettes or coding segments of the present invention. It can be used as a host cell for a vector. Two exemplary mammalian systems are: A COS cell line and a CHO cell line are used, respectively. Should be expressed in COS systems The vector containing the gene is transiently transfected into COS cells and The vesicles produce SV40 large T antigen constitutively. Vectors used for COS cells The vector typically contains the SV40 origin of replication, so that the vector is transfected into COS cells. When transfected, they replicate and the amount of protein expressed thereby To amplify.   CHO cells use the expression cassettes or coding segments of the present invention and Hydrofolate reductase or glutamine synthetase gene (the product of which (Which confer resistance) can be stably transfected with the vector . Cell lines with an increased number of constructs are used for increasing drug concentrations (eg, methotrexe ) In the cell line. Once selected, These strains are permanent reagents (which can be cryopreserved and Can be used to produce proteins whenever possible).   Code segment   The coding segment comprises a heterologous DNA coding site (complementary second heterodimer Can form α-helical coiled-coil heterodimer with buunit peptide A DNA region encoding a first heterodimeric subunit peptide) Then, a cleavage sequence is placed between the coding site and the DNA region. The cleavage sequence is the DNA region Region and in-frame, providing a target for chemical or enzymatic cleavage Encodes an amino acid sequence that Preferably, the cleavage sequence is that of the coil peptide. To facilitate its removal from its fusion partner (the selected polypeptide of interest) Included in the code segment of the present invention. However, the fusion peptide In applications where removal from the protein is not desired, the cleavage sequence may not be necessary. It is understood that there is no. In this regard, the present invention provides for heterologous DNA coding sites and complementary A second heterodimeric subunit peptide and an α-helical coiled coil A first heterodimer subunit peptide capable of forming A coding segment containing only the DNA region to be loaded is intended.   In a general embodiment, the coding site is a multiple cloning site (MCS) (Also called polylinker or polylinker site). MCS is a representative Contains the selected polynucleotide to be expressed in the coding segment or Numerous restriction enzymes useful for cloning into vectors containing Including elementary sites. Examples of suitable polylinkers include virtually any commercial cloning vector. (Eg, Stratagene, La Jolla, CA or Invitrogen, San Diego, CA From the vector).   In another embodiment, the coding site is a gene encoding the protein of interest. including. The generation of such a gene and its integration into the expression vector of the present invention Are described in Examples 9 and 10, respectively. In Example 9, the PAK ciliary antigen was The encoding gene anneals the synthetically produced complementary oligonucleotide And clone the ds fragment into the appropriate restriction sites in the pRLDE vector Generated. In Example 10, the cDNA encoding GFP was pRLDE Cloned in. Exemplary proteins that can be purified using the method of the invention As epoetin alfa, G-CSF, somatotropin, , TPA, urokinase and prourokinase, various interferons, EP O, as well as enzymes of various properties.   A coding segment is a coding region oriented either 5 'or 3' to a DNA region. May include positions. These two possibilities are illustrated in FIG. 6A, which shows a diagram of the expression cassette of the invention. And shown in 6B. The cassette is functionally functional in the chosen host cell system. Motor and, as described above, a coding sequence at the 3 'end of the promoter region. Is placed. 6A and 6B, the heterologous DNA coding site is "cDNA" And encodes the first heterodimer subunit peptide The DNA region is represented as "coiled DNA". FIG.6A is oriented at the 3 ′ end of the DNA region. Figure 6B shows the coding site oriented at the 5 'end of the DNA region. Show.   The cleavage sites that make up the target for rare-cutting proteases are Known in the art (Ausubel et al., 1988). Such proteases include: Factor Xa (Nagai and Thogersen, 1984, 1987) Thrombin (Smith and Johns on, 1988; Gearing et al., 1989), enterokinase (Dykes et al., 1988; LaVallie et al.). 1993), renin (Haffey et al., 1987) and collagenase (Germino and Bas). tia, 1984). Factor Xa and enterokinase are particularly useful . Because they cut at the carboxy terminus of their respective recognition sequences, Because it allows release of the fusion partner containing the true amino terminus of Ente The recognition sites for rokinase, factor Xa, and thrombin are No. 21, SEQ ID NO: 23, and SEQ ID NO: 25 are provided herein.   Providing a code segment having two different alignments of elements as described above The main reason for this is that the peptide (eg, coil peptide) Fusion with a polypeptide sometimes changes the desired activity or characteristics of the polypeptide of interest It is to make it. By generating two different fusions (one of which is Having the coil peptide at the N-terminus, and the other is selected for the coil peptide At the C-terminus of the polypeptide of interest), the desired The likelihood of obtaining a fusion that does not significantly alter the properties or characteristics of is increased.   Exemplary, DNA region encoding a first heterodimeric subunit peptide The coding sequences for SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 26, and SEQ ID NO: 27 Is an array represented by As shown in FIG. 6A (where the DNA region is Upstream of the coding site), the DNA region is typically as its first codon It is recognized that it contains an initiation ATG codon. The case as shown in FIG. Here, the DNA region is downstream of the coding site), and the start ATG codon is typically Provided by DNA encoding the polypeptide of interest inserted into the coding site .   The code segment of the present invention preferably comprises two alignments of elements as described above. Are provided in three arrangements. The three forms are the code part and the cut part Have different lengths of nucleotide spacers between Become. The spacer is used for the reading frame of the DNA region and the cleavage site. Provide code segments with coding sites in all three reading frames Included to provide. In this way, the appropriate coding segment is Regions and cleavage sites in-frame to match ends compatible with restriction sites present in MCS. It can be selected by those skilled in the art to express any DNA fragment having. V.Using heterodimeric subunit peptides for purification of fusion proteins Affinity matrix composition   The present invention also provides a tandem expression with a first heterodimer subunit peptide. Purify a polypeptide, such as a fusion protein, containing the selected polypeptide An affinity matrix composition that is useful for The matrix is The heterodimeric subunit peptide attached to it (this is the fusion tag Another complementary heterodimeric subunit pep expressed as part of the protein And a solid support having the ability to form an α-helical coiled coil). The affinity properties of the matrix depend on the selection of the complementary heterodimer subunit peptide. Based on alternative dimerization.   The selective dimerization process for purification is schematically illustrated in FIGS. FIG. 1A shows solid phase matrix 32 and heterodimer subunit peptide 34. 1 shows a selective dimerization matrix 30 of FIG. If necessary, matrix 32 , Is attached to peptide 34 via spacing group 36. The presence of such a group is Large or sterically bulky proteins to be purified on the column It can be particularly advantageous if it is a solid.   Solid phase matrix 32 is formed from any of a number of suitable materials known in the art. Can be done. Preferably, the solid phase material is packed in a chromatography column Spherical, such as aminopropyl-derivatized glass particles, which can form a deformed matrix Consisting of particles; however, the solid phase may be a continuous surface or non-spherical particles or beads. Can be formed. The method used for binding the peptide to the solid phase is Type, spacing group type (if present), and desired Can be easily determined by those skilled in the art based on the peptide bond of   The fusion protein is first prepared using methods known in the art (eg, Ausubel et al.). To obtain a suspension containing the fusion protein from the host cell expression system. Made. This suspension is typically used to prepare other fusion proteins in addition to the desired fusion protein. It is an impure solution containing impurities such as proteins.   Referring to FIG. 1B, such a fusion protein contains other proteins. For purification from impure solutions, the suspension must have an affinity matrix composition. When passed through an object, the affinity matrix 30 40, and a host complementary to the immobilized heterodimeric subunit peptide 34. It is logical to bind to fusion protein 38 containing rhodimer subunit peptide 42. Can be understood. Specifically interacts with the heterodimeric subunit peptide on the column Unbound components of the solution, such as non-working impurities 44, are also shown. These impure Debris is removed from the column environment by sufficient washing of the column .   Exemplary wash conditions include a pH between about 5.5 and about 8.0 (eg, 6.0). , And between about 0.1 M and about 1 M (eg, 0.5 M) of a salt (eg, NaCl). A salt washing step using an aqueous solution and a pH between about 5.5 and 8.0 (eg, 6.0). And between about 50% and about 100% (eg, 75%) of a suitable organic solvent (eg, , Acetonitrile) using an organic washing step. Appropriate Salts include sodium chloride (NaCl), potassium chloride (KCl), sodium acetate (NaOAc), ammonium chloride (NH_FourCl), ammonium acetate (NH_FourOAc), perchlorine Sodium salt (NaOClO_Four), Potassium perchlorate (KOClO)_Four), Sodium phosphate (N a_TwoHPO_Four), And potassium phosphate (K_TwoHPO_Four). A suitable organic solvent Are methanol (MeOH), ethanol (EtOH), isopropanol, Drofuran (THF), trifluoroethanol (TFE), and acetonitrile No.   The order in which the salt wash and the organic wash are performed is not important, but the organic wash first. If so, rinse the column thoroughly with an aqueous solution (eg, water) before beginning the salt wash. Thus, as much residual organic solvent as possible should be removed. For this reason, A salt wash is performed first, followed by an organic wash, followed by a final aqueous rinse, and Afterwards it is generally preferred to elute the desired protein from the column.   FIG. 1C shows an illustration of the column matrix after the washing step. Shown in FIG. 1B Impurities are no longer present in the column environment. Fusion protein 38 is now immobilized Heterodimer-subunit peptide 34 and complementary subunit 42 (this Is bound to the column via an interaction between ing.   1D and 1E-F show another example of eluting selected proteins 40 from a column. Means (ie, a means for releasing the selected polypeptide from the matrix) Stage). FIG. 1E shows that the fusion polypeptide 38 is removed from the column by coiled coil heating. Buffers that disrupt the interactions between the rhodimer subunits (eg, 2 shows a scheme eluted by washing with an aqueous buffer. Shown As such, fusion protein 38 is removed from the column by contact with such a buffer. Has been taken out. As described in detail below, a suitable ionic buffer is 0.5 M is guanidium chloride. Alternatively, the fusion protein has a pH of less than about 3.0 , And between about 20% and about 80% (eg, 50%) of a suitable organic solvent (eg, Tonitrile) can be removed by elution with a solution containing this The low pH of such an elution solution can result in the protonation of negatively charged residues (eg, Glu). And disrupt ionic interactions that stabilize the heterodimer. Another embodiment In another embodiment, the fusion protein has a pH greater than about 10.0 and has a pH of about 20% and about 80%. % (Eg, 50%) of a suitable organic solvent (eg, acetonitrile) Is eluted using a solution of The high pH of such elution solutions can lead to a positively charged residue. Ionic interactions that cause neutralization of groups (eg, Lys) and stabilize heterodimers For destroy.   To complete the purification process, the selected polypeptide 40 is subjected to enzymatic cleavage. By such a peptide cleavage reaction, the complementary heterodimer subunit peptide 42 (FIG. 1F).   Alternatively, the selected polypeptide 40 can be prepared in situ, such as by enzymatic cleavage. Is directly removed from the column. Cutting as illustrated in FIG. 1D Later, the polypeptide is ligated to the complementary subunit portion 42 (which is a coiled coil Column dimer as heterodimer subunit peptide 34 Remain bound) and split into selected proteins or polypeptides 40 Can be   From the above, it can be seen that the selective dimerization method of the present invention can Heterodimer subunit peptide capable of forming α-helical coiled coil Is a useful and efficient means for purifying fusion proteins containing Is understood.   Examples 8 and 12 are designed for purification of polypeptides including coiled peptides Describes the preparation and use of a modified affinity matrix, which comprises For attaching one of the heterodimeric subunit peptides to a matrix Exemplary Coupling Protocol, Application Using Derivatized Matrices Preparation of the affinity column, complementary heterodimer subunit peptide and / or Or through such a column as a fusion with the selected protein. Analysis of suspensions containing various contaminants along with peptides, as well as selected peptides Removal of the tide and / or fusion from the column.   Example 11 describes the Western blot of a protein purified using the method described above. The results of the analysis and ligand blot analysis are described. FIG. 16A shows a K coil The relative purity of the PAK (128-144) / E coil purified using the Shown is an image of a gel stained with Coomassie blue, as shown. As described in the examples The gel was blotted, and Western blot analysis and ligand blot Provided for analysis. The results shown in FIG. 16B show that the method of the present invention Can be successfully used to effectively purify proteins or polypeptides. You. VI.Detection reagent using heterodimeric subunit peptide   The present invention relates to the expressed proteins, in particular the α-helix sub Fusion protein having a heterodimeric subunit peptide capable of forming a unit Involves the synthesis and use of reagents to detect proteins. The reagent is a fusion protein Including but not limited to expression detection, ELISA applications, radioimmunoassays, etc. Not used in numerous research and clinical applications. The detection reagent is also selected May form part of a kit for detecting the expression of a given polypeptide. here, The kit may form an α-helical coiled-coil heterodimer according to the present invention. Expression vector containing a DNA region encoding a heterodimeric subunit peptide Also included.   In one aspect, the detection reagent is (i) designed and designed according to the methods described herein. And the formed heterodimeric subunit peptide, and (ii) a reporter component Including children. The reporter molecule couples to the heterodimer subunit peptide. (E.g., facing outward of the heterodimeric subunit peptide). At the heptad position (eg, position f), an amino acid coupling such as a cysteine residue To the coil peptide) Can be synthesized in a row (eg, as described in Example 10).   Alternatively, the reporter molecule can be a heterodimer, for example, as a radioactive amino acid. Can be incorporated into subunits. Radioactive amino acids are commercially available from a number of sources. Chemical and biological synthesis according to methods well known in the art. During the heterodimeric subunit peptide.   Reporter molecules include but are not limited to radioactive groups, fluorescent tags, enzymes, etc. May be selected from a number of reporter molecules well known in the art. Enzyme marker The use of and methods of incorporating them into biological detection systems are known in the art. (Eg, see Ausubel et al., (1988) for general methods; For illustrative exemplary reporter enzymes, see, eg, Lucif Chelase (Brasier et al., 1989; Gould and Subramani, 1988), chlorampheni Call acetyltransferase (CAT; Gorman et al., 1982), β-galactosida (An et al., 1982). Preferably, the reporter molecule is capable of forming a heterodimer. Non-interfering, attached to the coupling position in the heterodimeric subunit peptide Is done. One preferred position is position f of the subunit.   Example 13 describes a heterodimeric subunit peptide reporter according to the invention. Provides details for forming the molecule. FIG. 19 shows a protein expressed according to the present invention. In certain embodiments used to detect the presence of protein, the solid phase (ELISA 2) shows the reporter molecule in the assay.   In one exemplary embodiment, the reporter molecule is a heterodimer subunit. Knit fusion protein (eg, PAK cilia antigen / E coil fusion described in Example 9) ELISA) used to quantitatively detect the presence of Used.   FIG. 19 shows a 17 amino acid Pseudomonas aeruginosa strain formed from a peptide derived from PAK cilia. FIG. 1 shows a schematic diagram of an ELISA detection system for detecting a fusion protein to be formed. See figure In contrast, a solid phase, such as plate 50, can be prepared according to standard methods known in the art. Coated with a capture molecule such as PAK ciliary peptide specific antibody PK99H52. Fusion The sample containing protein 54 binds to the plate and binds the protein to antibody 52. Contacted under promoting conditions. As shown, fusion protein 54 is It consists of rhodimer subunit peptide 56 and PAK cilia peptide 58. Unbound After washing the plate to remove proteins, detection reagent 60 (this is a reporter -Comprising the complementary heterodimeric subunit peptide 62 attached to the molecule 64) Is added to the plate and with the complementary heterodimer subunit peptide 56 Reaction under conditions promoting the formation of a coiled-coil heterodimer complex between 62 and 62 Let me do. After further washing of the plate, detection of the reporter molecule is Used to quantify the amount of fusion protein 54 that is bound to.   Other embodiments of the invention using reporter molecules are described in the description provided above. It is clear. For example, the reagents can be used in complementary Used to detect the presence of a fusion protein containing a mer subunit peptide Similarly; the reagents can be obtained by plaque or by plating according to methods Can be used to detect expression of the fusion protein in a vesicle expression system.   The following examples illustrate the invention but do not limit it in any way. Not intended.                              Materials and methods   A.Abbreviation   The abbreviations used in the examples are t-BOC, tert-butoxycarbonyl; DCM, Chloromethane; DIEA, diisopropylethylamine; DCC, dicyclohexylcar Bodiimide (dicyclohexylcarbodimide); DMF, N, N-dimethylformamide; Gn d, guanidium; HF, hydrogen fluoride; DCU, dicyclohexylurea; BSA, bovine Serum albumin.   B.Buffer       Phosphate buffered saline (PBS)       10x stock solution, 1 liter:       80 g NaCl       2 g KCl       11.5 g Na_TwoHPO_Four・ 7H_TwoO       2g KH_TwoPO_Four       Working solution, pH 7.3:       137 mM NaCl       2.7 mM KCl       4.3 mM NaHPO_Four・ 7H_TwoO       1.4 mM KH_TwoPO_Four   C.Strains and plasmids   E. coli obtained from New England Biolabs, Beverly, MA. coli expression strain JM83 (Yanisch-Per ron et al., 1985) as host cells in experiments illustrating prokaryotic expression systems. did. Cells were harvested from Luria broth (LB) medium (MiMi) supplemented with 100 μg / ml carbenicillin. ller, 1972). Plasmids for eukaryotic systems were coli Top10F ' Grown and cloned in a strain (Grant et al., 1990) and Pichia pastoris GSl It was expressed in 15 strains (Cregg et al., 1985). Both of these are from Invitrogen (San Diego, CA).   Plasmid pASK40 (Skerra et al., 1991) was transformed into E. coli. Plasmid construction for E. coli expression Used for construction. The yeast expression plasmid pIC-9 (Invitrogen) was used for yeast expression. Used for plasmid construction.   D.Enzymes and oligonucleotides   Restriction enzymes and DNA modifying enzymes were purchased from GIBCO BRL (Gaithersburg, MD) . Deoxyribonucleotides were transferred to an automated DNA synthesizer Model 380A (Perkin-Elmer Appl. ied Biosystems Division, Foster City, CA). Plasmid DNA Separation and routine procedures were performed using standard procedures (Sambrook et al., 1989; Ausubel et al., 1988). ). Dideoxy sequencing of cloned plasmid constructs (Maxa m and Gilbert, 1977) with biotinylated primers and chemiluminescence detection. (Amersham, Arlington Heights, IL) using modified polymerase chain reaction (PCR ) Performed using the cycle sequencing protocol (Craxton, 1991).   E. FIG.SDS-PAGE   Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Using a 15% acrylamide gel, a mini gel device (“MINI-PROTEIN II” Dual Sl ab Cell, Bio-Rad Laboratories, Hercules, CA) in Laemmli et al. (1973). Performed as described. The gel was stained with Coomassie Blue (R-250, Bio-Rad) .   F.Western blot   Proteins on SDS-PAGE gels were analyzed using the protocol of Towbin et al. (1979) Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories) And transferred to a nitrocellulose membrane. Transfer is After 30 minutes, typically, under a constant current of 300 mA (Model 200 / 2.0 Power supply, Bi oRad) Completed.   Excess binding sites on the membrane are removed using 50 mM Tris-hydroxy-methylamino Tan (Tris) HCl (pH 7.5), 150 mM NaCl, 0.05% (v / v) "NONIDET-P40", 0.1% A chamber with a blocking solution consisting of 25% (w / v) gelatin and 3% (w / v) BSA Incubator shaker set at 100 rpm for 1 hour at room temperature (model G25  Gyroshaker, New Brunswick Scientific, New Jersey, USA). Blocked by incubation. 0.1% membrane at room temperature twice 10 mM Tris-HCl buffer (pH 7.5) containing "TWEEN-20" and 0.05% (w / v) BSA ) (TTBS).   G. FIG.Ligand blot   The proteins separated on the SDS-PAGE gel are transferred to a nitrocellulose medium as described above. Transferred to Memblen. Protein on the membrane, 1 membrane Time, PBS-ED buffer containing 6 M guanidine HCl (20 mM potassium phosphate (pH 7 .9), denatured by immersion in 200 mM KCl, 1 mM EDTA, 1 mM DTT). Ta The protein was slowly regenerated by four 1: 1 serial dilutions of the membrane in PBS-ED. And washed in fresh PBS-ED buffer to remove residual guanidine HCl. Me Membrane was added to 0.05% (v / v) Nonidet-P40, 0.25% (w / v) gelatin and 3% (w / v). / v) Incubate for 1 hour at room temperature in PBS-ED blocking buffer containing BSA. Blocked in beta shaker for 1 hour. Blot once with PBS-ED Sensation.                                 Example 1                         Chemical synthesis, purification and analysis   The peptide was synthesized by solid-phase peptide synthesis using benzhydrylamine-hydrochloride. Biosystems (Foster City, CA) Peptide Synthesizer Mod el 430A, as described previously (Hodges et al., 1988). It was chemically synthesized using xycarbonyl (t-Boc) chemistry. Peptide, resin Hydrofluoric acid containing 10% anisole and 2% 1,2-ethanedithiol (HF; 20 ml / g resin) for 1 hour at -5 ° C to 0 ° C. did.   The crude reduced peptide was subjected to reverse phase high performance liquid chromatography (RPC) and SYNCH ROPAK ”RP-P semi-preparation C₁₈Column (inner diameter 250 × 10 mm, particle size 6.5 μm, pore size 300 mm) ; Synchrom, Lafayette, IN) 0.5% B / min and 2 ml / min linear AB gradient Purified using Here, the solvent A is 0.05% trifluoroacetic acid (TFA) in water. And solvent B was 0.05% TFA in acetonitrile.   Amino acid composition and mass spectrometry were consistent with the designed sequence. Amino acid analysis The purified peptide was incubated in 6N HCl containing 0.1% phenol at 100 ° C. for 24 hours. The hydrolysis was carried out in a sealed tube evacuated for one hour at 160 ° C. or at 160 ° C. amino acid Analysis was performed on a Beckman model 6300 amino acid analyzer (Beckman, San Ramon, CA). Was. The exact primary ionic molecular weight of the reduced peptide was determined using the BIOION-20 Nordic (Uppsala, S Weden) was confirmed by a plasma desorption time-of-flight mass spectrometer.                                 Example 2                               Circular dichroism measurement   Circular dichroism (CD) spectra at 20 ° C, Jasco DP-500N data processor, (Model RMS) water bath (Brinkmann In) for temperature control of cuvettes and cuvettes Jasco J-500C spectropolarimeter equipped with instruments, Rexdale, Ontario, Canada asco, Easton, MD). Stationary N_TwoFlash was used. Reconnect equipment Calibration was routinely performed at 290 nm using an aqueous solution of crystalline d-10-(+)-camphor sulfonic acid.   The molar ellipticity at 200 nm is calculated as the average residue molar ellipticity ([θ]₂₂₀degcm^Two・ Dmol^{- 1} ) And is calculated from the following formula:       [θ] = [θ]_obs× mrw / 10 × 1 × c [θ]_obsIs the ellipticity measured in degrees, and mrw is the average residue molecular weight (amino acid residue Molecular weight of peptide divided by number), and c is in grams per milliliter Peptide concentration and 1 is the optical path length of the cell in centimeters. CDs The spectra were obtained by collecting data at 250 nm to 190 nm at 0.1 nm intervals. Was the average of the scans.   Peptide concentration was determined by amino acid analysis. pH was measured at room temperature.                                 Example 3                     Heterodimer versus homodimer formation   Peptides 0993 (SEQ ID NO: 26) and 0994 (SEQ ID NO: 27) were synthesized as described in Example 1. Were synthesized as follows. The first heterodimer subunit peptide 0993 (SEQ ID NO: 26 ) And a second heterodimeric subunit peptide 0994 (SEQ ID NO: 27) The CD spectrum of the peptide mixture at different ratios was measured as described in Example 2, The extent of heterodimer versus homodimer formation was determined.   Peptide was diluted with 0.1 M KCl and 50 mM potassium phosphate buffer (pH 7 at 20 ° C) (Reaction buffer). Total peptide concentration (0993 and 0994 The sum of the concentrations was 20 μM.   The data show that as the peptide ratio changes from 0: 100 to 50:50, the peptide Conformation changes from random coil structure to α-helix structure Indicates that you have done. An equimolar mixture of the 0993 and 0994 peptides is 220 and 208 nm At 220 nm, the average residue ellipticity at 220 nm is -31,760 deg^Two・ Dm ol^-1Which corresponds to about 100% of the α-helix structure (Hodges et al., 1990), This is because of the helical destabilization of homo-stranded coiled coils. Interionic ionic repulsion causes the formation of hetero-stranded coiled coils To provide the impetus for.   These results indicate that the mixture of peptides 0993 and 0994 To form a file.                                 Example 4     Attachment of peptide to heterodimer backbone by alkylation of thiol group   Binding to peptide sulphydryl groups was performed at ambient temperature at 50 mM N H_FourPerformed in OAc and 8M urea (pH 8). Bromoacetylation or iodoa The ceylated peptide was dissolved in buffer (0.987 μM, 2 ml) and peptide 0994 (SEQ ID NO: 27) was added (2 ml) to a final concentration of 0.165 μM. The reaction mixture is light It remains clear and is allowed to react at ambient temperature for 22 hours, at which point Acidified (pH 2) by careful addition of TFA and lyophilized.   A.Conjugate purification and identification   The reaction mixture (2 ml) was applied to a Synchropak RP-8 semiprep column (250 mm × 10 mm). I.D .; Synchrom Inc., Lafayette, IN). The conjugate was eluted by gradient elution (30 2% B / min over min, solvent A: 0.05% TFA / H_TwoO; solvent B: 0.05% TFA / acetoni Was easily separated from unreacted peptide using Freeze the isolated conjugate Dry and redissolve in HPLC grade water (200 μl), which is then Mono-S strong cation exchange column (Pharmacia, Uppsala, Sweden) I took it. The gradient used during this purification step was a 1% B / min gradient (solvent A: 5 mM NaH_Two PO_Four/ 20% acetonitrile (pH 5), solvent B: 5 mM NaH_TwoPO_Four/ 20% acetonitrile , 1M NaCl (pH 5)). The isolated conjugate is then applied to a reverse phase column and And desalted using a standard 2% B gradient (see above). Like this Yielded a pure conjugate, which was shown by mass spectrometry to be the desired product. (MW calculated: 7432.0, found, 7432.4).                                 Example 5                          Recombinant coil gene synthesis   The E coil gene was synthesized as follows. Highly expressed Pichi DNA sequence a codon bias based on the a pastoris gene (Koutz et al., 1989) Constructed from the back translation of the coil protein sequence designed Was. To avoid creating excessive restriction sites, four overlapping oligonucleotides And electrolysis on a 12% polyacrylamide gel containing 7M urea Purified by evacuation. The coiled DNA fragment was transformed into E as shown in FIGS. 5A to 5G. E. coli DNA polymerase I was synthesized using the Klenow fragment.   Oligonucleotides 1 (SEQ ID NO: 6) and 2 (SEQ ID NO: 7) Annealed together with the complementary sequences, and the remaining complementary strands were compared to FIGS. 5A and 5B. As shown in the above, synthesis was carried out using Klenow fragment. Use the same protocol for oligo Performed on nucleotides 3 (SEQ ID NO: 8) and 4 (SEQ ID NO: 9) (FIG. 5). D and 5E).   Next, the double-stranded fragment was digested with the restriction enzyme Alw21 I, and each fragment was digested. A sticky end was created at one end of the template (FIGS. 5C and 5F) and two flags Fragments were ligated with T4 DNA ligase to form a 177 base pair double-stranded fragment (FIG. 5). G). The fragments are cut out, electroeluted from the gel slice, and Digested simultaneously with EcoR1 and BamH1 and cloned into a plasmid vector Generated the appropriate 166 bp fragment.                                 Example 6                     E . Expression of coil constructs in E. coli   The expression vector pASK40 was modified at its polycloning site to De pRLD was generated. pASK40 was digested with EcoR1 and the resulting 5 ′ overhang was Smoothed with g-Bean Nuclease. The blunted plasmid was then digested with HindIII, And annealing oligonucleotides 5 (SEQ ID NO: 10) and 6 (SEQ ID NO: 11) Synthetic 43 bp annealed deoxyribonucleotide sequence Rejoined with Gment. This modification, which was confirmed by double-stranded DNA sequencing ) To shift the reading frame and to close the cloning site of pASK40. It was designed to more closely match the cloning site of the Pichia pastoris vector.   The synthetic E coil gene was double digested with EcoR1 and BamH1 as described above, and Forced cloning into pRLD digested with oR1 and BglII, coli E coil vector This resulted in the production of pRLD-E (FIG. 5H). Ligated plasmid in E. coli expression strain JM83 Were transformed by heat shock treatment (Yanisch-Perron et al., 1985) and Plated on selective LB plates supplemented with nisin (100 μg / ml). Coil remains Gene insertion was performed by restriction digest analysis followed by miniprep of successful transformants. Plasmid DNA was confirmed by DNA sequencing.   E. with pRLD-E. coli cells at 25 ° C with shaking at 100 μg / μl 0.5 A in LB medium containing benicillin₅₅₀Grown up to Expression is Ropyrthiogalactoside (IPTG) is added to a final concentration of 1 mM and added for 3 hours Induction was carried out by incubating at ℃ with shaking.   The expressed protein was subjected to modified osmotic shock treatment (Ausubel et al., 1988). I got it. Cells are harvested at 4000 xg for 10 minutes at room temperature and 5 mM EDTA and 20% screen 1 gram wet weight in 100 mM Tris-Cl (pH 8.0) (TES buffer) containing loin Resuspended at 80 ml per well. Cells were shaken at 200 rpm for 10 minutes at room temperature. Then The suspension is centrifuged again and the pellet is washed with 5 mM ice-cold MgSO_FourResuspended in (wet 80 ml per gram weight). Shake this on ice for 30 minutes, then 8000 xg for 4 Centrifuged at 10 ° C for 10 minutes. The supernatant constituting the periplasmic fraction was purified by the following Example 8 Further purification was performed using a coil affinity column, as described in.                                 Example 7                      Expression of coil constructs in yeast   The above E coil gene was ligated to the polymerase chain reaction (PCR; Saiki et al., 1988; Mullis 5 'EcoR1 site using as template for Mullis et al., 1987). And an E coil gene with a 3 'NotI site was generated. Synthetic Ecopri (sequence number No. 12) and Notpri (SEQ ID NO: 13) oligonucleotides were annealed at 50 ° C. 30 cycles at temperature were used as primers.   The PCR amplification product was transferred to plasmid pIC9 (Invitrogen, San Diego, Calif.) Containing the α-mating factor leader sequence for secretion into The chia E coil vector p9CE was generated. The successfully ligated plasmid was placed into E. coli. E. coli cloning strain TOP10F 'and transformed with LB / carbenicillin (100 μg / ml) on the plate. Prepare plasmid DNA from selected colonies And check for the correct insert size by digestion with restriction enzymes, And confirmed by DNA sequence analysis.   Identified yeast plasmids were prepared as described (Cregg et al., 1985) by Pichia pastoris. It was transformed into GS115 strain. GS115 is converted to YPD (1% peptone, 2% yeast extract, 2% % Dextrose) medium (Romanos et al., 1991) at 30 ° C. with shaking. 5 A₆₀₀And harvested by centrifugation. DH to cell pellet_TwoO and And 1 M sorbitol in water, followed by 25 mM EDTA and 100 mM dithiothreitol 1M sorbitol aqueous solution (SED buffer solution) containing A series of washes were performed. Release the final pellet to 10 mM sodium citrate. Buffer solution (pH 5.8) and 1 M sorbitol aqueous solution containing 1 mM EDTA (SCE buffer solution) ) And reconstituted with zymolase at a final concentration of 0.3 mg / ml Processed.   Spheroplasting was performed at 30 ° C. and aliquots taken at various time points were By treating with 5% SDS and checking its absorbance at 800 nm Monitored. Harvest cells by centrifugation at 70% spheroplasting And washed with 1 M aqueous sorbitol, 10 mM Tris-HCl (pH 7.5) and 10 mM CaCl_TwoWashing in 1M sorbitol aqueous solution (CaS buffer) containing Was. Resuspend the spheroplasts in CaS and use immediately for DNA transformation. Used.   In preparation for transformation, 10 μg of plasmid p9CE DNA was digested with the restriction enzyme BglII. To generate two linear fragments and run on a 0.7% agarose gel. Confirmed by electrophoresis. Freshly prepared spheroplasts are combined with digested DNA Incubated at room temperature for 0 minutes. 10 mM CaCl_TwoAnd 10 mM Tris-HCl (pH 7.5) Add a fresh solution of 20% PEG containing PEG (PEG / CaT) and incubate Continued for another 10 minutes.   Cells were harvested at 750 × g at room temperature and the supernatant was aspirated. Pellet the SOS medium (1M sorbitol, 0.3 × YPD medium, 10 mM CaCl_Two) And resuspend in cells Is allowed to recover for 20 minutes, after which 1M aqueous sorbitol is added and the selective RDB plate is added. Rate (1M sorbitol, 1% dextrose, 1.34% yeast nitrose) Base, 4 × 10-5% biotin and 0.005% amino acid (excluding histidine) ). Place the recombined clone in the absence of histidine Chromosomal AOX1 gene replacement by selective minimal methanol Ground (1.34% yeast nitrogen base, 4 × 10-5% biotin, 0.5% meta Nol). Successful linearization of plasmid fragment The clone after integration was used as a vector-specific primer (SEQ ID NO: 14, SEQ ID NO: 15) using PCR screening (Clare et al., 1991).   A recombinant yeast clone exhibiting a methanol-sensitive phenotype (MetS) was transferred to a BMGY medium (Pep Ton, yeast extract, yeast nitrogen base, biotin, glycerol) 1.0 A in₆₀₀Cultured until Next, the carbon and energy sources were changed to methanol (BMM Y medium) to allow induction using the AOX1 promoter (Ellis et al., 1985). After 72 hours of induction, the culture supernatant was collected by centrifugation at 4000 xg and And in the preparation for coil affinity purification described in Example 8 below. , Filtered through a 0.2 μm filter.                                 Example 8     Selective dimerization with affinity matrix affinity column                             Protein purification   This section describes an algorithm designed for the purification of polypeptides containing coiled peptides. The preparation and use of the affinity matrix is described. Affinity refined hands The order is the complement of the heterodimeric subunit peptide immobilized on the solid phase. Selective dimerization to proteins or peptides containing telodimer subunits based on. In particular, the polypeptide selected for purification may be an immobilized peptide. Designed and synthesized to contain a heterodimeric subunit peptide complementary to the The fusion peptide was   Generally, to prepare a selective dimerization affinity matrix, the phase One of the pair of complementary peptide subunits is linearized according to methods known in the art. Synthesized as a peptide, purified and then via one of the residues present in the peptide To bind to the solid matrix. Preferably, the cysteine residue is Available for matching.   The following sections describe the affinity to which the heterodimeric subunit peptide is attached. The preparation of the affinity matrix is described. Complementary heterodimer subunit pair Binding of peptide to affinity matrix and color of complementary subunit The conditions used for elution from the system are also detailed below.   A.Preparation of selective dimerization affinity matrix     1.Preparation of peptide. Design the complementary peptide as described above to Rix-coiled-coil heterodimers were formed. For binding to the solid phase, One of the peptide subunits contains a spacing group (C-terminal in SEQ ID NO: 27). Shown as the last 5 amino acids), and for chemical coupling to the matrix. Synthesized with a C-terminal cysteinamide for convenience.   The following complementary peptide pairs were designed and prepared as follows: SEQ ID NO: 26 (peptide 0993): Ac-EVSALEKEVSALEKEVSALEKEVSALEKEVSALEK-Amide SEQ ID NO: 27 (peptide 0994): Ac-KVSALKEKVSALKEKVSALKEKVSALKEKVSALKEGGGnLC-amide   gabcdefgabcdefgabcdefgabcdefgabcdef   Peptides were synthesized on a solid phase peptide synthesizer (430A Ap) using standard t-BOC chemistry. plied Biosystems Inc .; Foster City, CA). Peptide, fluorinated water It was cut off from the synthetic resin by treatment with acetic acid. Purification of each peptide by reversed phase high performance This was performed using liquid chromatography (RP-HPLC).   2.Coupling peptides to affinity matrices. Control hole (cont rol pore) aminopropyl glass resin (220 mg (22 μmol); Sigma, G-4643, flat Equilibrium: 500 angstroms, 200-400 mesh, amine content 110 umol / g gala Was washed twice with 5.0 ml each of DMF, DCM and DMF. Resin to 5.0 ml DIEA / Neutralized with DCM solution (5% v / v) for 5.0 minutes, then drained.   The resin was then stirred for 25 minutes in a solution of bromoacetic acid dissolved in a DCC / DCM solution . The DCC / DCM solution contains 138 mg (1.0 mmol) of bromoacetic acid (Aldrich Fine Chemicals Catalog No. 25.935-7) is dissolved in 2.0 ml of 0.5 M DCC / DCM solution and stirred for 15 minutes. And prepared by filtration to remove insoluble DCU. Resin again Drained and washed three times each with 5.0 ml of DCM, DMF, MeOH, DCM. Then get The activated glass resin obtained was dried.   Peptide 0994 (SEQ ID NO: 27; 26 mg, 6.15 μmol) was added to 3.0 ml of 100 mM NH 3_FourHCO_Three Dissolved in 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 5.0 ml of H_TwoO And washed.   The remaining unreacted bromoacetyl groups are_FourHCO_ThreeΒ-mercaptoethanol Cap by adding 5.0 ml of a 1% v / v solution of Stirred for 0 minutes. The resin is then drained and H_TwoWith O, DMF, and methanol Washed 5 times and then air dried for storage. Qualitative ninhydrin coupling Confirmed by test.   B.Preparation of affinity column   Prepared dimerization chromatography on RP-HPLC guard column (5mm x 20mm) -Resin was dry-packed. The column was then pumped at a flow rate of 0.2 ml / min for 10 minutes. Washed with the following solution: H_TwoO, 50% acetonitrile-H_TwoO, 50 mM potassium phosphate (PH 7.8) 5.0 M GndHCl and H_TwoO. This sufficient washing should be Performed to remove non-specific, non-covalently bound peptides as well.   C.Heterodimer subunits with selective dimerization affinity matrices Chromatography   Affinity columns prepared as described in detail in Part B above % H_TwoEquilibrated in O at a flow rate of 0.2 ml / min. 50 μl sample of peptide 0993 (distribution Column no. 26; 1 mg / ml in 10 mM phosphate buffer, pH 6.5) and eluted The fractions collected were collected for analysis by RP-HPLC. RP-HPLC, H_TwoFlat with 0.05% TFA in O Performed on an equilibrated C-8 Zorbax analytical column and dissolved at 2% / min at a flow rate of 1 ml / min. Elute with a gradient of liquid B (0.05% TFA acetonitrile) and detect at 210 nm did. After injection of the sample into the affinity column, the column is Le / H_TwoWash with a solution of O for 5.0 minutes and elute the eluate during the washing step by RP-HPLC. Collected for analysis. The peptide was then removed from the affinity column by 50 mM Solution of 5.0 M GndHCl in potassium phosphate (pH 7.8), followed by several volumes of H_TwoUse O And eluted.   FIG. 7A shows the chromatogram of peptide 0993 eluted from the RP-HPLC column in about 25 minutes. Show the system.   FIG. 7B shows the peptide coupled to the selective dimerization affinity column described in Part C above. After injection of tide 0993, the injection flow-through analyzed by RP-HPLC under the above conditions 2 shows the chromatogram of the minutes. The absence of a peak at 25 minutes indicates that virtually all Indicates that the loaded peptide was bound to the column.   FIG. 8 shows the chromatogram of the 60% acetonitrile washed fraction of the affinity column. Show the system. The presence of only the minor peptide peak at peptide 0993 position Indicates that the peptide was not eluted by the washing procedure.   FIG. 9 shows a chromatogram of the GndHCl eluted fraction. Major peak eluting in about 25 minutes The peak is peptide 0993, which is required for 0.5 M GndHCl to elute this fraction. It is effective. D.For the removal of non-specific peptides from a selective dimerization affinity column Condition   Studies were performed to remove nonspecific binding of charged peptides from the affinity column. In order to determine the conditions for this, we have carried out in support of the present invention. The following peptide Used in test mixtures. Peptide test mixture: Peptide sequence Net charge at pH 6.0 SEQ ID NO: 28 (Actin 1-28) Ac-DEDETTALVADNGSGLUKAGFAGADAPR-amide-4 SEQ ID NO: 29 (Anion exchange standard product) Ac-EYAAEAAEGLE-amide-4 SEQ ID NO: 30 (TnI 104-115) Ac-GKFKRPPLRRVR-amide +6   To test for non-specific binding, apply the affinity column to 10 mM phosphate Equilibration was performed in a buffer solution (pH 6.5) at a flow rate of 0.2 ml / min.   100 μl sample of peptide test mixture (1 mg / ml in 10 mM phosphate buffer, pH 6.5) Actin 1-28, 1 mg / ml anion exchange std., 2 mg / ml TnI 104-115) During the injection, the eluate was collected for analysis by RP-HPLC. Figure 10 shows the Affinity Chromatography of the peptide mixture that was run on the RP-HPLC column before loading on the tea column. 3 shows a mattogram. The conditions for flowing this column are detailed in Part D above. The conditions were the same as those described in (1). Shows the three above-mentioned peptides in the mixture Three major peaks are shown in the figure.   Next, the affinity column was washed with a solution of 0.2 M KCl / 10 mM phosphoric acid for 5.0 minutes. And during the washing step, the eluate was collected for analysis by RP-HPLC. FIG. The chromatogram of the eluted fraction is shown. A single major peak eluted at about 12 minutes is positive A (+6) positively charged TnI 104-115 peptide (SEQ ID NO: 28) is loaded on the column. Less retained, while both negatively charged proteins were retained. Show.   Next, the affinity column was washed with a 0.5 M KCl / 10 mM phosphoric acid solution for 5.0 minutes, During the washing step, the eluate was collected for analysis by RP-HPLC. Figure 12 shows the peptide Shows detectable peptide peaks eluting at positions corresponding to any component of the mixture Not.   Next, the affinity column was washed with a solution of 1.0 M KCl / 10 mM phosphoric acid for 5.0 minutes. And during the washing step, the eluate was collected for analysis by RP-HPLC. Shown in FIG. You The chromatogram shows the anion exchange standard peptide (SEQ ID NO: 29) and actin 1 This shows that both ~ 28 peptide (SEQ ID NO: 30) were eluted by this step.   Subsequent washes were performed with (a) 1.0 M KCl, and (b) 50 mM KCl._TwoPO_Four5.0M GndHCl in (pH7.8) Performed using the solution. These fractions were used for RP-HPLC analysis as described above. Collected. Figures 14 and 15 show that under these conditions, additional test peptide It was not eluted from the ram.   E. FIG.Purification of recombinant PAK pilin / E coil peptide   This section describes the purification of a recombinant PAK pilin / Ecoil fusion protein. The fusion protein was produced as described in detail in Example 9.     1.Preparation of a peptide affinity matrix.1 g (110 μmol) of amino Nopropyl control pore glass resin (Sigma, Cat♯G-4643) in 15.0 ml DMF, DC Washed twice with M and DMF. Resin was washed with 15.0 ml of 5% v / v DIEA / DCM solution Neutralized for minutes and then drained. 2 ml of 0.5 M dicyclohexylcarbodiimide / D The CM solution was added to 138 mg (1.0 mmol) of a 0.5 M dicyclohexylcarbodiimide / DCM solution. Was added and the mixture was stirred for 15 minutes.   The mixture was then filtered, and the prepared glass resin beads were added to the solution. The final volume of the mixture was made up to 20 ml with dichloromethane. Stir the mixture for 25 minutes And then discharged. Wash resin 3 times with 15.0 ml DCM, methanol and DCM And then dried. Qualitative ninhydrin test to confirm coupling Went for. 100 mg (23.7 μmol) of peptide 0994, 30.0 ml of 100 mM NH_FourHCO_Three( pH 8.0) and then added glass resin beads. 30 minutes of peptide resin solution While stirring. The resin is then drained and 5.0 ml of H_TwoWashed with O. Unremaining The bromoacetyl group in the reaction was replaced with 100 mM NH_FourHCO_Three1% v / v β-mercaptoe in pH 8.0 Cap by adding 20.0 ml of the ethanol solution to the resin, and mix the mixture for 15 minutes. Stirred for minutes. The resin is then drained and H_TwoO, 0.1% TFA, H_TwoO, ethanol , Washed 5 times with acetone and then dried for storage. Amino acids Removed for analysis and theoretical tolerance determination.     2.Preparation of affinity column.Two columns for affinity purification I) Stainless steel RP-HPLC guard column (5mm x 20mm) and   ii) Stainless steel RP-HPLC column (10 mm x 200 mm). Prepare each column Dry-packed to the top with the obtained dimerization chromatography resin. packing After that, each column was washed with the following solution at a flow rate of 0.5 ml / min. H_TwoO, 50m 5.0 M GndHCl in M potassium phosphate solution (pH 7.0), 50% acetonitrile containing 0.1% TFA Lil / H_TwoO, and H_TwoO.     3.Purification of recombinant peptides. The column is first treated with 10 mM phosphate buffer (pH 6.0). Adjusted. Filter the periplasmic fraction or whole cell extract at a flow rate of 0.2-0.5 ml / Loaded in minutes. After loading, replace the column with 5.0 ml (small column) or 20 ml (large Column) 0.5 M KCl in 10 mM phosphate buffer (pH 6.0), followed by 10 mM phosphate buffer Washed with 80% acetonitrile (v / v) in liquid (pH 6.0). Of the bound peptide Final elution was performed with 50% acetonitrile / H with 0.1% TFA._TwoWith O (v / v / v) wash went.   The presence of the recombinant protein in the eluted fraction was determined by RP-HPLC (Zorbax C-8 analytical column (10 m m × 250 mm)) using a linear AB gradient of 1.0% B / min from 0% to 60% B Evaluation was performed at a flow rate of 1.0 ml / min. Here, the solvent A is 0.05% trifluoroacetic acid in water. (TFA) and solvent B was 0.05% TFA in acetonitrile. All implementation The time was 60.0 minutes.   FIG. 17 shows a crude periplasm containing a load fraction containing recombinantly expressed E-coil peptide. From the loaded sample and the affinity column 3 shows an RP-HPLC chromatogram of the eluted fraction. The HPLC run is shown as follows A: a. Before elution; b. Crude periplasmic extract, c. Periplasmic extract bray Break-through; d. 0.5M KCl wash; e. 80% acetonitrile wash F. First elution wash; g. A second elution wash; h. Breakthrough re-pass and First elution wash after subsequent wash.   FIG. 18 shows recombinantly expressed PAK-cili-E coilope derived from crude periplasmic extract. Figure 4 shows an RP-HPLC chromatogram of the purification of the peptide. All HPLC runs were performed as described above. I went to. The HPLC runs are shown as follows: a. Before elution; b. Coarse periplasmic extraction Birth, c. Breakthrough of periplasmic extract; d. 0.5M KCl wash; e. 80 % Acetonitrile wash; f. First elution wash; g. Second elution wash.                                 Example 9                        PAK Ciliary antigen / E coil fusion   17 amino acid ciliated epithelial binding domain of PAK cilia (SEQ ID NO: 5) (128-144; Lee et al., 1994) encoding two complementary oligonucleotides (oligonucleotide sequences). SEQ ID NO: 16 and SEQ ID NO: 17) were synthesized. Sterilize equimolar ratio of oligonucleotide Dissolve in water, mix together, heat to 80 ° C. for 5 minutes and slowly cool to room temperature This was annealed. 4% agarose annealed oligonucleotide Gel electrophoresis and excise 54 bp fragment and modified freeze compression It was eluted from gel slices by the freeze-squeeze method (Sambrook et al., 1989). Simple To put it simply, the excised band was applied to a spin filter column (BioRad.Richmond .CA) and placed in liquid nitrogen for 5 minutes. Then, eppen the filter Into a dorf tube and in a microcentrifuge at maximum speed (about 14,000 rpm ) Rotated.   After ethanol precipitation, the eluate was treated with T4 polynucleotide kinase at 37 ° C for 30 minutes. And inactivated the enzyme at 65 ° C. for 15 minutes. Then the phosphorylated fragment Was cloned into shrimp alkaline phosphatase treated EcoR1 digested pRLDE And transformed into E. coli strain JM83, pRLD on LB / Carbenicillin plates. An E-PAK clone was generated.   Insert orientation and sequence, restriction analysis and miniprep DNA plasmid Was confirmed by sequencing. Expression and purification of PAK / E coil fusion protein , E coil proteins as described previously.                                 Example 10                    TheLean fluorescent protein / E coil fusion   A green fluorescent protein (GFP) cDNA clone (Prasher et al., 1992) was cloned from ClonTech (P alo Alto, CA). GFP-specific PCR primers (oligonucleotide sequence No. 18 and SEQ ID No. 19) were constructed with flanking EcoR1 restriction sites and GFP Gene PCR was performed under standard conditions (Saiki et al., 1988). 1% agar for PCR product Run on a roth gel and excise the 700 bp amplification product and run on a gel as described above. Eluted.   Plasmid pRLDE-GFP digests the eluted DNA fragment with EcoR1, and Ligate DNA into shrimp alkaline phosphatase treated EcoR1 digested pRLD-E It was built by doing. Next, pRLDE-GFP was transformed into the E. coli TOP 10F 'strain. Orient the gene in an LB / CA / IPTG transformation plate (100 μg / μl carbenicillin, 0.8 Determined by direct detection of fluorescent colonies on (mM IPTG). Gene sequence Confirmed by restriction analysis and sequencing. Protein expression and purification Performed as described in.                                 Example 11     PAK / Western and ligand blot analysis of E-coil fusions   From E. coli JM83 cells containing expressed Ecoil-tagged PAK17 mer peptide Crude periplasmic preparation and purified by K-coil affinity column The PAK17 / E coil was transferred to a power supply model 1420A (BioRad Laboratories) for 200 minutes. Fractionation was performed by SDS-PAGE at a constant voltage of V. Figure 16A, stained with Coomassie blue The image of the gel is shown. Lanes are as follows: Lane 1: Crude PAK (12 8-144) / Ecoil periplasm preparation; lane 2: K coil affinity color Lane 3: Rainbow marker (2- 45 kDa).   The gel was blotted and subjected to Western blot as described above. Monoclonal elicited against Pseudomonas aeruginosa K strain cilia (PAK cilia) Antibody PK99H was diluted with TTBS (1: 500) and 1 hour at room temperature with gentle shaking Incubated with blot. The blot is then washed three times with TTBS and Goat anti-mouse IgG (H + L) -alkaline phosphater diluted 1: 10,000 with TTBS Conjugate (Jackson Laboratories) was incubated with the blot as described above. I did it.   The blot was washed three times with TTBS, followed by a final wash with Tris-buffered saline. Antibody binding was performed using alkaline phosphatase substrate (100 mM NaCl and 5 mM MgCl_TwoContains Nitro blue tetrazolium chloride dissolved in 100 mM Tris-HCl (pH 9.5) And 5-bromo-4 chloro-3-indolyl phosphate). And visualized by: For color development, remove the nitrocellulose strip with deionized water. Stopped.   Ligand blots act as probes for biotin-labeled (bt) K coils Performed to evaluate ability to do. The gel shown in FIG. 16A was blotted as described above. And biotin-labeled K coil (1 mg / ml) with PBS-ED blotting buffer 1: 500 and mix with blot with gentle agitation at room temperature for 1 hour. Incubated. The membrane was washed three times (10 minutes per wash).   Streptavidin-alkaline phosphatase conjugate (Jackson ImmunoResear ch Laboratory) with PBS-ED blocking buffer (1: 2500; vol / vol) Then incubated with the blot for 1 hour at room temperature with gentle agitation. The blot was washed three times with PBS-ED, and the substrate buffer (100 mM Tris-HCl (pH 9.5), 100 mM NaC l and 5 mM MgCl_Two). K-coil binding is performed by Toroblue tetrazolium chloride and 5-bromo-4-chloro-3-indo Visualization was performed by adding ril phosphate (NBT / BCIP). Color development, Nitrose The lulose strip was stopped by rinsing with deionized water.   The results of Western blot and ligand blot analysis are shown in FIG. 16B. Leh Two to three and eight to nine were probed using the ligand blot technique. on the other hand , Lanes 5-6 and 11-12 were probed using Western blot technology. Was. Lanes 1, 4, 7, and 10 are size markers (rainbow markers; 2-45)  lane 2: coupled to crude PAK (128-144) / Ecoil periplasm preparation Lane 3: purified by K-coil affinity column Lane 5: Crude PAK (128-144) / bt K coil bound to PAK (128-144) / E coil Lane 99: Monoclonal antibody, PK99H, binding to Ecoil periplasm preparation : K Binds to PAK (128-144) / E coil purified by Ilaffinity column Monoclonal antibody, PK99H; Lane 8: K-coil affinity column Bt K coil binding to purified E coil; lane 9: Bt-K binding to PAK cilia Coil; Lane 11: E coil purified by K coil affinity column Monoclonal antibody that binds, PK99H; Lane 12: Normal mouse that binds to PAK cilia IgG.                                 Example 12      Isolation of PAK / E coil fusion using K coil affinity column   A.Isotope ⁽¹⁵ N) labeling   Single colony of E. coli JM83 strain with recombinant expression plasmid (pRLDE-PAK) Was inoculated into 5 ml of LB medium containing 100 μg / ml carbenicillin (Sigma) and incubated at 37 ° C. And grown overnight. Overnight culture (2 ml) was transferred to 200 ml of mini medium (0.6% NaH_TwoPO_Four, 0. 3% K_TwoHPO_Four, 0.05% NaCl, 4 mM MgSO_Four, 2μM FeSO_Four, 0.1%^FifteenNH_FourCl (Cambridge Isotope Laboratories, Woburn, MA) and 1% D-glucose (pH 7.2). I was inoculated. Once OD₆₀₀Reaches 0.8-1.0, the IPTG Was added to the culture to a final concentration of 1 mM. The culture was grown for an additional 6 hours at 37 ° C. For further purification.   B.E.coli Preparation of periplasm preparation   The induced gene expression culture was collected by centrifugation (4000 × g, 10 minutes) . Pellet the pellet with 100 ml of TES buffer (100 mM Tris, 10 mM EDTA and 20% sucrose). And incubate for 10 minutes at room temperature with gentle agitation. Was. Cells are harvested by centrifugation (7000 × g, 10 minutes) and 5 mM MgSO 4_FourAgain Suspended. The suspension was incubated in an ice-cool bath for 30 minutes with stirring . Centrifuge the supernatant containing the periplasmic protein (5,000 xg, 10 minutes). Collected and stored at -20 ° C or lyophilized.   C.PAK / HPLC affinity column for purification of E coil   Lyophilized expressed crude periplasmic preparation was added to 20 ml of 10 mM phosphate buffer (pH 6). Dissolved. Wash the lysed sample (5 ml) with 10 mM phosphate buffer (pH 6.0). After equilibration, equilibrate the affinity column with the K-coil peptide. I did it.   The constant flow rate for HPLC was 0.2 ml / min. Remove the column after loading the sample. Washed by injecting 5 ml of 0.5 M KCl in phosphate buffer (pH 6) for 5 minutes. 5 ml of 80% acetonitrile in 10 mM phosphate buffer (pH 6.5) for 25 minutes after washing with 0.5 M KCl Was injected. The PAK / E coil fusion was transformed with ddH containing 0.1% TFA_Two50% acetonitrile in O Eluted with 5 ml of ril. Identification of eluted samples is determined by reversed-phase chromatography and quality Confirmed using quantitative analysis.                                 Example 13                                 Detection reagent   This section describes the complementary peptides and α-helical coiled coils that are compatible with the present invention. Fusions with heterodimeric subunit peptides capable of forming heterodimers Describes the synthesis and use of reagents for detecting proteins.   A.Iodination reagent   Heterodimer subunit peptides were formed as described above. Then, The peptide was iodinated according to standard procedures known in the art. Preferably, yo Heterodimer subunit peptides designed for iodination are tyrosine residues However, iodination also includes, for example, the Bolton-Hunter reagent (which is preferentially Lysine residues). For example, the subunit anti Lysine occupying position f of the repeat unit can be so derivatized. Bolton-Hunte Kits containing the reagents are commercially available (eg, ICN, Costa Mesa, CA).   Another iodination method is the [IODOGEN] method. Here, 2 mCi of carrier-free Na^{1 twenty five} I, 75 μl of 0.5 M phosphate buffer (pH 7.4) and 20 μl of 1 μg / μl peptide Add to μg “IODOGEN” coated polypropylene test tubes. Chu The solution was stirred for 8 minutes and the solution was washed with a 10 x 0.46 cm C-8 reverse phase Chromatography (Brownlee Labs. Santa Clara, CA) by HPLC. . Sample material is reduced from 0.1% trifluoroacetic acid to 60% in 0.1% trifluoroacetic acid. Elution was performed with a gradient to acetonitrile. Primary radiation-active iodinated peptide Is detected by gamma detection of the fraction and the reporter in the method of the invention. Used as a molecule.   B.Enzyme binding reagent   Enzyme binding reagents can be made according to methods well known in the art. One such In the method, a desired protein such as alkaline phosphatase or luciferase The gene for the porter enzyme is changed to the gene for the heterodimer subunit The fused and expressed fusion protein is complemented with a complementary protein according to the method described above. Affinity on rhodimer subunit peptide affinity column Purified by chromatography.   In another method, a heterodimeric subunit peptide is converted to a reporter enzyme. Chemically bond. For example, horseradish peroxidase (HRP) is a peptide Present in the subunit (preferably located at the f heptad position of the coil peptide Cysteine residues) via a disulfide bond. One way In the above, HRP (6 mg / ml 0.1 M sodium phosphate buffer (pH 7)) was added for 0.5 to 1 hour. Of the crosslinking reagent (N-succinimidyl 4- (N-maleimide) at 30 ° C. Methyl) cyclohexane-1-carboxylate (SMCC) or N, N-dimethyl N-succinimidyl 6-maleimidohexanoate (S) dissolved in formamide MH) (10% solution). The reacted HRP was mixed with Sephadex G-25 (0.1M Equilibrated with phosphate (pH 6); by gel filtration of Pharmacia, Piscataway, NJ) Isolate. Reactive HRP with reduced cysteine containing heterodimer subunit For coupling, the two components were mixed in a 1: 1 molar ratio and containing 2 mM EDTA. In a 0.1M sodium phosphate buffer (pH 6) at a final concentration of 0.01 to 0.15mM at 4 ° C for 20 hours Or for 1 hour at 30 ° C. The binding product is “ULTROGEL” AcA 44 (Pharmacia Bio tech, Piscataway, NJ).   The HRP-conjugated heterodimer subunit peptide has 4- When reacted with aminoantipyridine and the increase in absorbance at 510 nm is measured, Acts as a reporter molecule.   Although the invention has been described with respect to particular methods and embodiments, various modifications and adaptations may be made. It is understood that changes and modifications can be made without departing from the invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12N 1/21 C12N 1/21 C12P 21/02 C12P 21/02 // (C12P 21/02 C12R 1:19) (72) Invention Yu, Ray United States of America California 92037, La Jolla, Via Alicant 3141-A (72) Inventor Bautista, Daisy Canada T6G 2S5 Alberta, Edmonton, 112T Street NH. Wu. 1B-9105 (72) Inventor Hodges, Robert S. Canada T5G 2B2 Alberta, Edmonton, Saskatchewan Drive 9045 (72) Inventor Trippet, Brian Canada T6H 5B5A Alberta, Edmonton, Mickner Park 304-2 (72) Inventor Katya, Paul Jay. Canada Tee 6G 0S4 Alberta, Edmonton, 81 Este Treat 11040 (72) Inventor Irvine, Randall T. Canada Country Tea 8 G 1 Sea 1 Alberta, Sherwood Park, Chelsea Mayner 9

Claims (1)

[Claims] 1. Appropriate expression vectors for expressing the heterologous protein in the host cell. Code segment for use in the following:   (I) a heterologous DNA coding site,   (Ii) complementary second heterodimer-subunit peptide and α-helix First heterodimer-subunit capable of forming a coiled-coil heterodimer A DNA region encoding a peptide, and   (Iii) a cleavage sequence located between the coding site and the DNA region, wherein the DN is In-frame with A region, containing target for chemical or enzymatic cleavage Cleavage site encoding amino acid sequence Where:   (A) one of the first and second heterodimer-subunit peptides is g comprising at least two heptad amino acid repeats in the form of abcdef, wherein At positions a and d of each amino acid repeat are leucine, isoleucine, and Positions e and g of each amino acid repeat are selected from the group consisting of Selected from the group consisting of paraginic acid and glutamic acid,   (B) the other of the first and second heterodimer-subunit peptides is g It contains at least two heptad amino acid repeats in the form of 'a'b'c'd'e'f'. Where the positions a ′ and d ′ of each amino acid repeat are leucine, isoleucine, And valine, and positions e and And g are selected from the group consisting of lysine, arginine, and histidine; hand   (C) in the complementary heptad of the α-helical coiled-coil heterodimer Each corresponding d / a ′ and a / d ′ pair has one residue valine and the other Consists of residues selected from the group consisting of leucine and isoleucine, Code segment. 2. Multi-cloning wherein said coding site is capable of inserting a heterologous DNA coding region 2. The segment of claim 1, comprising a site (MCS). 3. The segment of claim 1, wherein the coding site comprises a heterologous DNA coding region. G. 4. The DNA region comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 26, and SEQ ID NO: 27 2. The sequence of claim 1 having a sequence selected from the group consisting of: Segment. 5. 2. The cleavage sequence of claim 1, wherein the cleavage sequence encodes an enterokinase cleavage site. Segment. 6. A second polypeptide comprising a selected polypeptide having functional activity and complementary Heterodimer-subunit peptide and α-helical coiled-coil heteroda A first heterodimer-subunit peptide capable of forming an imer is selected from the selected A fusion protein linked to the N-terminus or C-terminus of the polypeptide obtained,   here   (A) one of the first and second heterodimer-subunit peptides is g comprising at least two heptad amino acid repeats in the form of abcdef, wherein At positions a and d of each amino acid repeat are leucine, isoleucine, and Positions e and g of each amino acid repeat are selected from the group consisting of Selected from the group consisting of paraginic acid and glutamic acid,   (B) the other of the first and second heterodimer-subunit peptides is g It contains at least two heptad amino acid repeats in the form of 'a'b'c'd'e'f'. Where the positions a ′ and d ′ of each amino acid repeat are leucine, isoleucine, And valine, and positions e and And g are selected from the group consisting of lysine, arginine, and histidine; hand   (C) in the complementary heptad of the α-helical coiled-coil heterodimer Each corresponding d / a ′ and a / d ′ pair has one residue valine and the other Consists of residues selected from the group consisting of leucine and isoleucine, Fusion protein. 7. One of the first and second heterodimer-subunit peptides is a Having a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 26, and The peptide has a sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 27 The fusion protein of claim 6, wherein 8. A reagent for detecting the presence of an expressed polypeptide, comprising:   Complementary first heterodimer-subunit peptide and α-helical coil Second heterodimer-subunit peptide capable of forming a coiled heterodimer And wherein the first heterodimer-subunit peptide is Second heterodimer-subunit peptide expressed in tandem with the polypeptide Tide, and   A detectable report bound to the second heterodimer-subunit peptide Tar And where   (A) one of the first and second heterodimer-subunit peptides is g comprising at least two heptad amino acid repeats in the form of abcdef, wherein At positions a and d of each amino acid repeat are leucine, isoleucine, and Positions e and g of each amino acid repeat are selected from the group consisting of Selected from the group consisting of paraginic acid and glutamic acid,   (B) the other of the first and second heterodimer-subunit peptides is g It contains at least two peptide amino acid repeats in the form of 'a'b'c'd'e'f'. Where the positions a ′ and d ′ of each amino acid repeat are leucine, isoleucine, And valine, and positions e 'and G 'is selected from the group consisting of lysine, arginine, and histidine; hand   (C) in the complementary heptad of the α-helical coiled-coil heterodimer Each corresponding d / a ′ and a / d ′ pair has one residue valine and the other Consists of residues selected from the group consisting of leucine and isoleucine, reagent. 9. A method for detecting expression of a selected polypeptide, comprising the following steps:   A step of expressing the fusion protein according to claim 6 or 7,   The fusion protein comprises a second heterodimer-subunit peptide and the same. Contacting it with a detection reagent consisting of a detectable reporter bound to it Wherein the first heterodimer-subunit peptide is contacted with the The second heterodimer-subunit peptide and α-helix under the conditions of the process A process capable of forming a coiled-coil heterodimer; and   Detecting the presence of the heterodimer A method comprising: 10. The detection reagent comprises the second heterodimer-subunit peptide and a tag. Claims: Consisting of a fusion protein comprising a green fluorescent protein in an undem 10. The method according to 9. 11. One of the first and second heterodimer-subunit peptides is Having a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 26, and Has a sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 27. The method of claim 9, wherein 12. A kit for detecting the expression of a selected polypeptide, comprising:   (I) an expression vector,   A replication segment that allows the vector to replicate in a selected host cell; And   In the 5'-3 'direction, the following:   (A) a promoter functional in the host cell, and   (B) The code segment according to any one of claims 1 to 5, wherein The coding segment is a DNA region adjacent to the coding site or the promoter An expression cassette comprising a coding segment, which can be oriented with any of An expression vector; (II) a detection reagent,   The first heterodimer-subunit peptide is tandem with the polypeptide. Said second heterodimer-subunit peptide expressed in the   A detectable report bound to the second heterodimer-subunit peptide Tar A detection reagent A kit comprising: 13. Affinity matrix composition for purification of selected polypeptides Thing,   A solid support, and   First Heterodimer Subunit and α-Helix Coiled Coil Heterodide A second heterodimer-subunit pair attached to the support, capable of forming a dimer Pseudo, Including   Here, the selected polypeptide is the fusion protein according to claim 6 or 7. An affinity matrix composition that is part of the substrate. 14． The affinity according to claim 13, wherein the solid support is a glass resin. -Matrix composition. 15. A method for purifying a selected expressed polypeptide, comprising: :   (I) in a suitable host cell expression system, the first heterodimer subunit Expressing a fusion protein containing the selected polypeptide in tandem ;   (Ii) obtaining a suspension containing the fusion protein from the host cell expression system;   (Iii) the suspension is   (A) a solid support, and   (B) an alpha helicopter composed of the first and second heterodimer subunits Forming said fusion protein by forming a coiled-coil heterodimer The first heterodimer under conditions that promote immobilization of the first heterodimer to the matrix composition. The α-helical coiled-coil heterodimer with the subunit A second heterodimer-subunit peptide bound to a solid support, Passing through an affinity matrix composition, comprising:   (V) the fusion protein comprises the α-helix coiled-coil heterodimer The affinity matrices remain bound to the affinity matrix via A step of washing the ricks, wherein the washing step is performed in any order by: :   (A) having a pH between about 5.5 and 8.0 and containing between about 0.1 M and about 1 M salt; Salt washing process comprising washing the affinity matrix with a solution And   (B) having a pH between about 5.5 and about 8.0, and between about 50% and about 100% of the organic solvent; Washing the affinity matrix with a solution containing a medium. Organic washing process, A process comprising:   (Vi) releasing the selected polypeptide from the matrix; A method comprising: 16. 16. The method of claim 15, wherein said expression system is an E. coli or yeast expression system. . 17． The releasing step has a pH of less than about 3.0, and comprises about 20% and about 80% Eluting the fusion protein with a solution containing an organic solvent between 16. The method of claim 15, comprising. 18. The releasing step has a pH greater than about 10.0, and comprises between about 20% and about 80%. Eluting the fusion protein with a solution containing an organic solvent during The method of claim 15, wherein 19. The organic solvent is methanol, ethanol, isopropanol, Selected from the group consisting of drofuran, trifluoroethanol, and acetonitrile 16. The method of claim 15, wherein the method is selected. 20. The method according to claim 19, wherein the organic solvent is acetonitrile. 21. The salt is sodium chloride, potassium chloride, sodium acetate, ammonium chloride; Ammonium, ammonium acetate, sodium perchlorate, potassium perchlorate, sodium phosphate The method according to claim 15, wherein the substance is selected from the group consisting of thorium and potassium phosphate. the method of. 22. 22. The method according to claim 21, wherein said salt is NaCl. 23. One of the first and second heterodimeric subunit peptides is Having a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 26, and The peptide has a sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 27 The method of claim 15, wherein 24. A kit for purifying a selected polypeptide, comprising:   (I) an expression vector,   A replication segment that allows the vector to replicate in a selected host cell; And   In the 5'-3 'direction, the following:   (A) a promoter that is functional in the host cell, and   (B) The code segment according to any one of claims 1 to 5, wherein The coding segment is a DNA region adjacent to the coding site or the promoter An expression cassette comprising a coding segment, which can be oriented with any of An expression vector;   (II) an affinity matrix composition,   A solid support, and   The first heterodimer subunit peptide and α-helical coiled coil A second heterodimer-sub attached to the support capable of forming a heterodimer Unit peptide,   An affinity matrix composition comprising:   Including   Wherein the selected polypeptide is such a first heterodimer sub A kit which is part of a fusion protein also comprising a unit. 25. Further comprising a host cell suitable for use with the expression vector. The kit according to claim 24.