JPH10510155A - Method for producing peptide using recombinant fusion protein construct - Google Patents

Abstract

  (57) [Summary] Methods for isolating and / or purifying recombinant peptides by using a fusion protein construct comprising a carbonic anhydrase and a variable fusion polypeptide are provided. The method comprises precipitating either the fusion protein construct or a fragment of the fusion protein construct comprising carbonic anhydrase. Also provided are inclusion bodies containing the fusion protein construct, and methods for producing peptides that include expressing the fusion protein as part of the inclusion body. Fusion protein constructs comprising carbonic anhydrase and a specific target peptide are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION             Method for producing peptide using recombinant fusion protein construct                                 Background of the Invention   Developments have been made on in vitro DNA manipulation and the transfer of accompanying genetic information, Techniques to enable efficient expression of endogenous and foreign proteins in microbial hosts It has become a nology. Recombinant DNA methods are involved in protein and peptide expression Selection, amplification and manipulation.   Expression of all foreign proteins in any microbial host is theoretically possible. However, the stability of the proteins produced has often limited such practice, Yield has been incurred. Specifically, small foreign proteins and oligonucleotides are mostly It is not easily overproduced in minute cell hosts. Small pep in host cells Tide expression increases the likelihood that the host will assimilate the polypeptide.   In response to this problem, small peptides have been developed using peptide markers (eg, beta- Galactosidase or chloramphenicol acetyltransferase) It is expressed as a fusion protein with a second large peptide. The resulting fusion protein White is not easily assimilated, but isolation and purification of the desired protein is often very efficient Ineffective or ineffective.   Column chromatography separation, for example, separation by affinity column One that allows the isolation and / or purification of recombinant peptides that do not require use The development of a method would be particularly desirable. Typically, such columns are expensive and have May have a relatively limited lifetime. Therefore, isolation of the recombinant peptide And there is a continuing need for flexible and convenient methods for purification You.                                 Summary of the Invention   The invention includes carbonic anhydrase and variable fusion polypeptide A method for producing a recombinant peptide using a fusion protein construct is provided. Typically , Carbonic anhydrase is a mammalian carbonic anhydrase (mC A). Carbonic anhydrase and variable fusion polypeptide The peptides can be linked together by a cleavage site. The cleavage site is Or an amino acid sequence that is selectively cleaved by treatment with a cleavage agent It produces things. Cleavage agents are specific chemical cleavage sites, such as methionine residues. It may be a chemical reagent that recognizes the position, for example, cyanogen bromide. Cleavage agents are specific Specific points on amino acids or amino acid sequences ("enzymatic cleavage sites") May be an endopeptidase that cleaves the construct in the process.   One embodiment of the present invention comprises precipitating a fusion protein construct, comprising: Oriented to the manufacturing method. The precipitated construct can be resolubilized. After cleavage, Fragments containing carbonic anhydrase ("carbonic anhydrase" Lase fragment ”) by chromatography, filtration, or Alternatively, it can be removed by a precipitation method.   A second embodiment is (i) cleaving the fusion protein construct to produce a soluble carbonic anhydride. Obtaining a drase fragment and a soluble variable fusion polypeptide fragment And then (ii) precipitating the carbonic anhydrase fragment A method for producing a peptide is provided. Carbonic anhydrase fragment The fragment contains carbonic anhydrase and contains other amino acid residues, e.g., cleaved Derived from the interconnecting peptide produced by the reaction or It may include those derived from chemical residues. Variable fusion polypeptide fragment Contains at least one copy of the target peptide and has an N-terminal or C-terminal tail. Additional amino acids such as rows or intraconnecting peptides It may contain a noic acid residue. More than one variable fusion polypeptide fragment When containing many copies of the target peptide, typically the target peptide Are linked by an internal binding peptide containing   A third embodiment of the invention is directed to a host cell, for example, an E. coli host cell. Expressing the fusion protein construct as part of an inclusion body in The present invention provides a method for producing a peptide, comprising isolating a product. Typically, fusion proteins The white construct contains at least two copies of the target peptide. However, The combined protein constructs were GRF (1-44) (SEQ ID NO: 20), GLP1 (7-34). ) (SEQ ID NO: 21) and PTH (1-34) (SEQ ID NO: 22) Single copy if the target peptide corresponding to the more selected peptide is included Construct containing only the target peptide can be expressed as part of the inclusion body You. GRF (1-41) (SEQ ID NO: 23), GLP1 (7-34) (SEQ ID NO: : 21) and PTH (1-34) (SEQ ID NO: 22) are growth hormones, respectively. Screening agents that respond to glutazone-releasing factor, glucagon-like peptide 1 and parathyroid hormone Represents a peptide having a amino acid. Amino acids 1-44 (G RFR (1-44) (SEQ ID NO: 20)) and amino acids of glucagon-like peptide 1 The sequence for noic acid 1-36 (GLP1 (1-36) (SEQ ID NO: 24)) is Application No. PCT / US94 / 08125, the disclosure of which is incorporated by reference. It is deemed as described herein. Human and parathyroid hormone (PTH (1 -84) (SEQ ID NO: 25)) is the sequence related to amino acids 1-84 of Hendy et al., Proc. Natl. Acad. Sci., USA, 78, 7365 (1981) and T. Kimura et al., BBRC, 114, 493 ( 1983), the disclosure of which is incorporated herein by reference. Consider   Another embodiment of the present invention provides for the cleavage of a fusion protein Obtaining a drase fragment and a variable fusion polypeptide fragment And a method for producing a peptide. Carbonic anhydrase fragment And the variable fusion polypeptide fragments are precipitated. Fragmented sediment Extract with a solvent containing organic components to obtain a variable fusion polypeptide fragment. Can be recovered.   The present invention also provides a fusion protein expressed in a host cell, for example, an E. coli host cell. An inclusion body comprising the white construct is provided. The fusion protein construct is a carbonic anhydrer And target peptides. Preferably, the target peptide is GRF (1-41 ) (SEQ ID NO: 23), GLP1 (7-34) (SEQ ID NO: 21) and PTH (1 -34) An amino acid corresponding to a peptide selected from the group consisting of (SEQ ID NO: 22) At least one copy of the amino acid sequence.   Finally, the present invention provides a fusion comprising a carbonic anhydrase and a target peptide. Synthetic protein constructs are also provided. The target peptide was GRF (1-41) (SEQ ID NO: 2). 3), GLP1 (7-34) (SEQ ID NO: 21) and PTH (1-34) ( SEQ ID NO: 22) contains the amino acid sequence corresponding to the peptide selected from the group consisting of No.                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows a variable fusion polypeptide formed from multiple units of a target peptide. Here are various formulas for C.                             Detailed description of the invention   Linkage between the recombinant vector and a biosynthetic device called a host cell or host organism Expression of foreign proteins is a well-known method for biochemical protein synthesis. Departure Akira has identified a new, low-cost, and efficient method for large-scale biological synthesis of peptides. Combine with a carbonic anhydrase-based fusion protein construct to establish The novel modification of this expression method attached is used. The method of the present invention is based solely on chemical methods. Process, i.e., affinity chromatography or other For isolation and / or purification of recombinant peptides without the need for a chromatography step Process design for Also, fusion protein constructs may be used, if desired. Allows the use of Gand immobilized affinity separation methods. The method of the present invention is expensive Eliminate machinery and reagents, long synthesis times, low reaction efficiencies, and solid-phase peptides Fewer incomplete copies than the synthetic method, resulting in higher yields of the resulting peptide.   The method of the present invention facilitates the expression of foreign proteins by biological systems, Including combinations of cellular factors and biological components. The expressed fusion protein structure Since the structure contains the binding protein carbonic anhydrase, expression of cells or organisms It does not allow for the disruption of specific identity to the target peptide by the mechanism. preferable The inducible expression mechanism is based on the peptide construct (which otherwise would be toxic to host cells). (Which may have the property).   The method of the present invention also provides factors that substantially contribute to the efficiency, capacity and yield of the purification process. including. Including carbonic anhydrase and carbonic anhydrase The solubility properties of the fusion construct are distinctive of the variable fusion polypeptide from other components. Facilitates complete separation. Relatively low molecular weight carbonic anhydrase Allows for increased capacity and efficiency per unit weight of the construct. The method is particularly No additional reagents or reactions are required, thus avoiding the formation of unwanted by-products You.   The size of the fusion protein construct varies with the nature and copy number of the target peptide Will be. The fusion protein construct is large enough to avoid degradation by host cells (Eg, at least about 60 to 80 amino acids), depending on the host cell. Not so large that they cannot be effectively expressed. In practice, The fusion protein construct will have a molecular weight of up to about 500,000, Constructs are also within the scope of the present invention. The fusion protein construct is expressed by a host cell and Choosing the size of the fusion protein construct to avoid introducing errors in white sequences I do. This is a consequence of the number of target peptide copies present in a given construct. It imposes practical restrictions. Range of restrictions imposed by the factors discussed above Within, the actual number will vary depending on the size and nature of the individual target peptides. Would.Carbonic anhydrase   Carbonic anhydrase is derived from various sources. Suitable source is spine An animal, particularly of mammalian origin, for example, human carbonic acid Hydrolase (hCA), rat carbonic anhydrase, cat carboni Cook anhydrase, equine carbonic anhydrase or bovine carboni Cook anhydrase. Goats, rats, pigs and other mammals such as monkeys Carbonic anhydrase from a mammalian species may also be used to form the fusion protein construct of the present invention. I Can be Examples of suitable carbonic anhydrases include human carbonic acid. Quanhydrase II (hCAII; Taylor et al., Biochemistry, 9, 2638 (1970 )).   Carbonic anhydrase is a modified functional version of carbonic anhydrase. It may be a lase. The modified function version depends on the solution conditions, for example, the specific p H, or carbonic acid that can precipitate as a function of metal ion or salt concentration It will retain the ability of inhydrase. Between about 3.2 to about 6.0, more Preferably, by adjusting the pH of the solution to between about 3.5 and about 5.5, Modified versions that can precipitate from solution are preferred. Typically, the modifier version Is the inhibitor benzenesulfonamide, acetoazolamide or their derivatives It also retains the ability of carbonic anhydrase to bind strongly to. Sulfani Of carbonic anhydrase for luamide and acetoazolamide inhibitors A detailed discussion of the binding properties can be found in International Application PCT / US91 / 04511. And are deemed to be as described herein by reference.   Examples of suitably modified carbonic anhydrases include (I) methionine. (II) that all or some glutamic acid has another negatively charged amino acid; (III) all of which are substituted by a carboxylic acid, preferably aspartic acid, Means that some arginine is converted to another positively charged amino acid, preferably lysine (IV) all or some asparagine is replaced with another amino acid (V) methionine is substituted with another amino acid, preferably substituted with glutamine Acid, preferably by alanine, serine, cysteine, threonine or leucine. Or (VI) cysteine is replaced with another amino acid, preferably A functional substitution variable replaced by phosphorus, threonine, leucine or alanine Includes variants.   An example of a suitably modified version of carbonic anhydrase is hCAI Polypeptides exhibiting functional fragments of carbonic anhydrase such as I Cysteine 205 of hCAII, asparagine 231, methionine C-terminal truncation or proline in 240 or leucine 250 21 or N-terminal truncations at glycine 25, including but not limited to . Functional fragments are those that precipitate from a wide range of solutions by adjusting the pH. Retains the ability of rubic anhydrase.   Another appropriately modified version is a substitution modification of hCAII with the following substitutions: Includes variants and substitutional variants of functional fragments of hCAII:   i. All or some glutamic acid residues are separated by another negatively charged amino acid ( AA), hCAII preferably substituted by an aspartic acid residue   ii. All or some arginine residues are another positively charged AA, preferably Or hCAII substituted by a lysine residue   iii. [N11X, G12X] (asparagine, glycine), [N62X, G 63X], [N321X, G232X], M240X (methionine), or C With one or more modifications to the AA position at 205X (cysteine) HCAII or a functional fragment thereof having the following modifications These AA positions are present in certain fragments): Asparagine changes to glutamine, or glycine changes to alanine, Nin changes to alanine, serine, cysteine, threonine or leucine, and And cysteine changed to serine, threonine, leucine or alanine.   Preferably, carbonic anhydrase and carbonic anhydrase A fusion protein comprising between about 3.2 and about 6.0, preferably no more than about 3.5 Adjust the pH of the solution to between about 5.5, more preferably between about 4.0 and about 5.0. Can precipitate from a wide range of solutions. Carbo using this method Examples of solutions in which nick anhydrase can precipitate are polyethyleneimine and urea (eg, For example, at least 2M) or guanidine hydrochloride (eg, at least 2M). Solution. Preferably, the carbonic anhydrase has no more than about 3.2 However, outside the pH range of about 6.0, it remains soluble in a wide range of solutions.Variable fusionpeptide   Variable fusion polypeptides may have several forms (eg, FIG. 1). reference). The first contains a single target peptide. The second is amino acids or Are multiple side-by-side units of a single target peptide linked by an internal binding peptide. Consists of In this case, the internal binding peptide is usually (Linking carbonic anhydrase and variable fusion polypeptide) Although different in structure and selectivity, they are not necessarily different. Strange Where the internal binding peptide is nevertheless the same general as the interconnecting peptide Two different types of enzymes that have enzymatic or chemical properties Cleavage agents separately cleave variable fusion polypeptides from carbonic anhydrase And will cleave the individual target peptides together. The third form is Are connected side by side by an inner connecting peptide Several (ie two or more) identical or different markers It is a single unit composed of a target peptide. The fourth form is an internal binding peptide. Consists of multiple side-by-side units connected by tides And each unit is different, further linked by an internal binding peptide Contains the same series of individual target peptides. The fifth form is an internal binding peptide Consists of the same series of side-by-side units linked by Each unit is furthermore identical to several identical units linked by an internal connecting peptide. Or different target peptides, which are not repeated for each unit. The sixth mode is composed of a plurality of identical side-by-side units. The knit is further comprised of several identical target pairs linked by an internal connecting peptide. Including peptides.   The target peptide may be a natural or synthetic peptide desired as a product, e.g. Contains all or part of the desired protein, oligonucleotide or small peptide You may. For the purposes of this application, peptides are linked by peptide bonds Contains at least two amino acid residues. Single or variable in the variable fusion polypeptide Suitable specific examples of target peptides that may appear as multiple linked units include Cartolin, calcitonin, insulin, tissue plasminogen activator, Growth hormone, growth hormone releasing factor, erythropoietin, interferon, Interleukin, oxytocin, vasopressin, ACTH, collagen binding Protein, parathyroid hormone, glucagon-like peptide, glucagon, proinsulin , Tumor necrosis factor, substance P, brain natriuretic peptide, antibody Heavy and light chains, respectively, in particular Lerner, Science, 246, 1275 et seq. Individual antibody chain fragments, such as isolated variable regions as characterized by (VH or VL) and E. Ward et al., Nature, 341, 544-546 (1986). Encompasses epitope regions as more characterized (where antibodies, chains, Fragments and regions contain the natural antigenicity or immunogenetics of the antigenic material With antigenicity obtained in a specific manner). Further specific examples of desirable polypeptides are sweetened. Taste peptide, tranquilizing polypeptide, nerve growth factor, regulatory protein, functional form Enzyme, DNA polymerase, DNA modifying enzyme, structural polypeptide, neural peptide Pide, cardiovascular system, respiratory organs, excretory organs, lymphoid organs, immunity, blood, reproduction, cell stimulation Polypeptides, leukemia inhibitors, anti- Bioactive peptides and bacteriostatic peptides (cecropin, atasin, apidaesin) Including insecticidal, herbicidal and fungicidal peptides, and lysozyme You.   formula: -TargP- (CS2)-[-(Ln1)_n-(CS1)_m-TargP- (C S2)-]_r [Wherein -CS1- and -CS2- are cleavage sites,-(Ln1)-is a linking sequence, -TargP- is the target peptide, n and m are 0 or 1, r is 1 to about 150 Means an integer up to] A peptide comprising an amino acid sequence having the formula: belongs to a suitable variable fusion polypeptide . The target peptides were GRF (1-41) (SEQ ID NO: 23), GLP1 (7-3) 4) corresponding to (SEQ ID NO: 21) or PTH (1-34) (SEQ ID NO: 22) Amino acid sequence. -(CS1)-and-(CS2) -cleavage The site may be a chemical or enzymatic cleavage site. Enzymatic cleavage The position can be recognized by endopeptidase or exopeptidase (eg, , When r is 1,-(CS2)-is the C-terminal residue of the construct). Preferably, the enzyme Goal opening The cleavage site is recognized by endopeptidase.Cleavage site   The fusion protein construct may have a chemical or enzymatic cleavage site. The cleavage site or sites that may be included in the fusion protein construct are Depends on the identity of the target peptide present. Typically, the target peptide is cleaved Cleavage site and target peptide are selected so that the amino acid sequence corresponding to the position is not included I do. Next, consider the selection of individual cleavage sites. In some cases, enzymatic cleavage The cleavage site may be designed to avoid the use of a reaction. S-cyanylating agent (example For example, after treatment with 2-nitro-5-thiocyanatobenzene) or about 3.0 PK less than_aUse of a chemical cleavage site that can be cleaved by treatment with an acid having And this can be done by: In other examples, a special functional group, for example, Allows modification of the target peptide to introduce a C-terminal α-carboxamide group It may be desirable to use a unique cleavage site.   Chemical and enzymatic cleavage sites and peptides in close proximity to one of these sites Corresponding agents used to effect bond cleavage are described in International Application PCT / US91 / 04511 and PCT / US94 / 08125, which are incorporated herein by reference. These disclosures are considered to be set forth herein by reference. In the present invention A peptide sequence suitable for use as a cleavage site (and encoding DNA gene sequences) and their corresponding cleavage enzymes or chemical cleavage conditions. It is shown in Table 1 below. The indicated gene sequence is one of the ones encoding the corresponding peptide sequence. It is possible. Other DNA sequences encoding the same peptide sequence may be constructed . Production of recombinant peptides   Transformed with an expression vector carrying the recombinant fusion protein construct gene The host cell can be used to express the fusion protein construct. Prokaryotic host cells Alternatively, it may be a eukaryotic host cell or a cell in a higher organism. One preferred of the present invention In certain embodiments, the host cell is a microbial host cell, such as E. coli. Fusion protein Insert the DNA segment encoding the construct into the appropriate underlying vector To prepare a vector carrying the protein purification construct gene Can be. International application for expression of single and multiple copy recombinant fusion protein products PCT / US91 / 04511, which is hereby incorporated by reference. Are considered to have been   Expression vectors are used for recombinant genes as well as for transcription, translation, phenotyping, Post-expression processing of expression regulation or other expression regulation, RNA binding and expression products Including basic vector segments, such as suitable regulatory DNA sequences for I have. In general, promoters, operators, regulatory sequences and transcription termination signals Structural features such as null are contained in the expression vector. Host cells or higher life Expression vectors from any of the basic vectors that are compatible with the May be synthesized. The regulatory sequences of the expression vector may be prokaryotic or It will be adapted or applied to eukaryotic host cells or higher organisms. Polypeptide structure Post-expression regulatory sequences that cause the secretion of the construct may be included in the eukaryotic expression vector. No. The expression vector has a stimulatory effect on host cells or higher organisms, resulting in In particular, the polypeptide construct is overexpressed over the normal biosynthetic expression of the host. preferable.   Typically, the form of a host cell or higher organism using the resulting recombinant vector The recombinant gene is inserted into a suitable base vector used for the transformation. Host details As a product soluble in cells or higher organisms, or as a product insoluble in cells. To express the fusion protein construct. Alternatively, the fusion protein construct May be expressed as a product secreted by the host cell or higher organism. Good More preferably, inclusion bodies, that is, aggregates of insoluble substances, are formed in host cells such as E. coli. one The fusion protein construct may be expressed as a part. Fusion expressed as part of inclusion bodies Synthetic protein constructs typically include two or more copies of the target protein. Construction of most single-copy fusion proteins based on carbonic anhydrase Have been found to be expressed as soluble products. For example, hCAII And a single copy of the target peptide (eg, calcitonin, substance P, Geotensin, ATPase-IP, εSU ATPase and asparagine A single copy fusion protein construct containing a synthetase Is expressed. Surprisingly, however, in some cases, One copy of a carbonic anhydrase-based fusion protein is part of the inclusion body It has been found that it can be expressed as In detail, hCAII and single copy GRF (1-41) (SEQ ID NO: 23), GLP1 (7-34) (SEQ ID NO: : 21) or PTH (1-34) (SEQ ID NO: 22) was obtained from E.co. li BL21 F^-ompTrB^-mB^-Part of inclusion bodies in E. coli host cells such as (DE3) Is expressed as   The present invention relates to a fusion protein construct or fusion protein comprising carbonic anhydrase Precipitate any of the construct fragments (collectively, "CA-based proteins"). And a method for producing a recombinant peptide. hCAII is It is known that a nearly neutral solution of its intact form of the enzyme precipitates when acidified. Have been. Lowering the pH removes zinc cations from intact enzymes, It is thought to induce qualitative conformational changes and cause precipitation. Surprisingly, carbonic anhydrase, in particular, such as hCAII Milk carbonyl anhydrase, whether in intact form or not, It has been found to exhibit this same solubility behavior as a function of pH. That In a similar manner, fusion proteins containing carbonic anhydrase as a function of pH It has been found that white can be precipitated. Typically, the pH is from about 3.2 to about 6. 0, preferably between about 3.5 and about 5.5, more preferably between about 4.0 and CA-based proteins can be easily precipitated from solution by adjusting to between about 5.0 Can be made. The CA-based protein is converted to a high salt fertility solution, for example, at least About 5% (w / v) sodium sulfate or at least about 5% (w / v) sulfuric acid It may be precipitated from an ammonium-containing solution.   In one embodiment of the present invention, a method comprises precipitating a fusion protein construct. Thus, the recombinant peptide is isolated. Fusion protein structures expressed as soluble products A precipitation step is performed on the fusion protein construct expressed as part of the building or inclusion body. I can. In the latter case, prior to the precipitation step, for example, at least about 0.5. Dissolve inclusion bodies in a solution containing 5M citric acid. Fusion protein constructs can be Solutions, eg, chaotropic agents (eg, guanidine hydrochloride) or surfactants (eg, , SDS), a solution containing at least about 2 M urea, or It may be dissolved in a solution having a pH of at least about 10.0.   The pH of the solution is between about 3.2 and about 6.0, preferably between about 3.5 and about 5.5. In time, the fusion protein construct can be precipitated from the solution. You. In some cases, addition of salt to the solution other than pH adjustment may improve precipitation efficiency. Can be up. Suitable salts are sodium chloride, potassium chloride, magnesium sulfate Includes sodium, sodium sulfate, sodium acetate and lanthanum chloride. Mg²⁺, Ca²⁺, Zn²⁺, Sr²⁺Or Co²⁺PH adjustment of divalent metal ions such as Precipitation efficiency may be improved in combination with nodes. Add the solubilized fusion protein construct to When precipitating from a lukali solution, typically acetic, formic, citric, phosphoric and phosphoric acids are used. And the pH of the solution is adjusted by adding an acid such as hydrochloric acid. By adjusting pH Alternatively, a sufficient amount of sodium or ammonium sulfate By adding such a salt, the solubilized fusion protein construct may be precipitated. .   In some cases, as an initial step in isolating the recombinant peptide, It may be useful to remove cell debris and nucleic acid material from crude cell lysates Absent. Typically, at least about 0.25%, preferably at least about 0.38% This is done by adding polyethyleneimine (PEI) to the lysate. In general, the addition of PEI will result in about 90-95% of the cell debris and DNA present. Precipitate. Then, adjust the pH of the PEI solution to between about 3.5 and about 6.0. MCA-based fusion protein can be easily precipitated from the supernatant .   For example, have a pH of at least about 10, preferably at least about 10.5 By dissolving in a solution, the precipitated fusion protein can be redissolved. Sinking Transferring the fusion protein to a chaotropic agent, a detergent or at least 0.5M. It may be redissolved in another type of solution, such as a solution containing citric acid. After cleavage, The fragment containing the rubic anhydrase ("carbonic anhydrase Fragmentation) is removed by chromatography, filtration or, preferably, precipitation. You can leave. As mentioned above, the pH of the solution is between about 3.5 and about 6.0. Easy precipitation of carbonic anhydrase fragments by readjustment Can be made. Alternatively, if desired, the ligand immobilization affinity Using a separation method, the carbonic anhydrase fragment is separated from the cleavage product. May be separated. For example, a solution of the cleavage product is converted to a benzenesulfonamide compound or Are inhibitors that include acetoazolamide compounds (eg, affinity columns) By contacting it with an intact or denatured form of carbonic anhydrase Lagment can be removed. Preferably, a benzenesulfonamide compound The product is an amino-substituted benzenesulfonamide (eg, 4-amino-benzene) Sulfonamide) or its derivatives I do. The acetoazolamide compound is 5-acetamido-1,3,4-thiadiazole -2-sulfonamide or a derivative thereof.   In another embodiment of the present invention, a method for producing a recombinant peptide comprises: (i) The fusion protein construct is cleaved to yield a soluble carbonic anhydrase fragment and And soluble variable fusion polypeptide fragments are obtained, and then (ii) Precipitating the nick anhydrase fragment. Carbonic Ann The hydrolase fragment contains other amino acid residues besides carbonic anhydrase. It may contain a group. Typically, these other amino acid residues are It is derived from an interconnected peptide present in the building. Suitable interconnects for use in the present invention Peptides include amino acid sequences that include a chemical or enzymatic cleavage site. cleavage Later, typically, the carbonic anhydrase fragment is an interconnected peptide Or at least a part of a derivative thereof. Generally, the derivative is cleaved Generated as a by-product of the reaction. For example, the C-terminal peptide bond of a methionine residue Has a C-terminal homoserine residue by cleaving the fusion protein construct in May produce carbonic anhydrase fragments   Supernatant fraction remaining after precipitation of carbonic anhydrase fragment Can be isolated by centrifugation. Supernatant fraction is variable fusion poly Contains peptide fragments and, if desired, used for peptide isolation The variable fusion polypeptide fragment is further purified by conventional methods.   Precipitated to improve recovery of variable fusion polypeptide fragments Extraction of carbonic anhydrase fragments with solvents containing organic solvents Good. Suitable solvents are acetonitrile, propanol, citric acid, polyethylene Contains organic components such as glycols, or mixtures thereof. For example, organic solvents , For example, 50% acetonitrile aqueous solution and 50% n-propano The extraction can be performed using an aqueous solution of water.   Before subjecting to the cleavage reaction, the fusion protein construct may be purified by various methods. For example, using a ligand-immobilized affinity separation method or a precipitation method The fusion protein construct can be purified. Requires regeneration or affinity purification Who rely only on wet chemical separation methods such as precipitation, filtration or centrifugation If the recombinant fusion protein construct is obtained by the Purification of the construct is preferred.   A third embodiment of the present invention provides a method for constructing a fusion protein construct in an E. coli host cell with a portion of an inclusion body. As a recombinant protein, and then isolating the fusion protein construct. A method for producing a tide is provided. Typically, the fusion protein construct will contain multiple copies of the target Contains peptide. However, as described above, one mCA as well as GR F (1-41) (SEQ ID NO: 23), GLP1 (7-34) (SEQ ID NO: 21) And a single copy of amino acids corresponding to PTH (1-34) (SEQ ID NO: 22) It is possible to express a fusion protein construct comprising the sequence. Sealed in E.coli host cells Examples of suitable single copy fusion proteins that can be expressed as a host are hCAII and A single copy of GRF (1-41) (SEQ ID NO: 23) and GRF (1- 44) (SEQ ID NO: 20), GLP1 (1-34) (SEQ ID NO: 26) GLP1 (7-34) (SEQ ID NO: 21), GLP1 (7-36) (SEQ ID NO: 27), GLPI (7-37) (SEQ ID NO: 28), PTH (1-34) (SEQ ID NO: 2) 2), PTH (1-38) (SEQ ID NO: 29) and PTH (1-84) (sequence No. 25).   Isolating inclusion bodies from crude cell lysates by conventional methods, such as centrifugation Can be. 50 mM Tris, 1 mM EDTA, pH 7.8 solution or 1 Cell debris and / or nuclei present by washing the inclusion bodies with 00 mM EDTA solution The crude inclusion body can be subjected to an initial purification step, such as to remove acid material.   The inclusion bodies can be dissolved under various conditions. For example, a pH of less than about 3.2 A solution under acidic conditions (eg, containing at least about 500 mM citric acid) Solution). Alternatively, a pH of at least about 10 The inclusion body may be dissolved in a solution having A small amount of N-lauroyl sarcosine When the surfactant is added to the solution, dissolution of the inclusion body under basic conditions Can be easier. An effective amount of 6N HCl or urea or N-lau Loylsarcosine, sodium dodecyl sulfate, sodium octyl sulfate or Solution containing surfactant such as tiltrimethylammonium bromide (CTAB) The inclusion body may be dissolved in the liquid. After dissolving the inclusion body in the solution as described above, Adjust the pH to between about 3.5 and about 6.0, preferably between about 4.0 and about 5.0. Surprisingly found that solubilizing fusion protein constructs I did. Alternatively, methods involving affinity separation of ligand immobilization Alternatively, the solubilized fusion protein construct may be isolated.   Another embodiment of the present invention provides for the cleavage of a fusion protein Obtaining a drase fragment and a variable fusion polypeptide fragment; A carbonic anhydrase fragment and a variable fusion polypeptide Including settling both of the fragments. The precipitated fragment is The variable fusion polypeptide fragment can be recovered by extraction with a solvent containing You. Suitable solvents are acetonitrile, propanol, citric acid, polyethylene glycol. Contains organic components such as recalls or mixtures thereof.   The present invention is further described by reference to the following detailed examples.Embodiment 1 FIG. Description of expression system Host cell origin, phenotype and genotype   E. coli BL21 F^-ompTr_B ^-m_B ^-(DE3) from Novagen, Inc., Madison , Obtained from WI. These E. coli cells harbor the bacteriophage T7 promoter. High level expression of the cloned gene in an expression vector containing the gene. T7 RN A bacteriophage (DE3) containing the A polymerase gene was converted to BL21 (DE3 ) Integrated into the chromosomal DNA of the cells. The T7 RNA polymerase gene is la c Controlled by UV5 promoter and lacI gene product.Construction of pBN1   The expression vector pET31F1mhCAII containing the hCAII gene was Obtained from Dr. P.J.Laipis of Lorida University. Tanhauser et al., Gene, 117, 113 (1992) The pET31F1mhCAII was prepared as described by Step Rasmid pET31F1mhCAII is a plasmid in pUC-derived plasmid backbone. HCAII downstream of the Teriophage T7 promoter (human carbonic acid) Hydrase II). Two synthetic oligonucleotides Creotide 5'-A GCT TTC GTT GAC GAC GAC GA T ATC TT-3 ′ (SEQ ID NO: 7) and its complementary sequence 5′-AGC T AA GAT ATC GTC GTC GTC AAC GAA-3 ′ (sequence No. 8) was cloned into pET31F1mhCAII digested with HindIII. I got it. This plasmid was designated as pA1.   Plasmid pA1 was digested with restriction enzymes SspI and BspEI and the resulting ends Was smoothed by T4 DNA polymerase treatment. T7-hCAII cassette DNA fragment from the pA1 digest containing pBR322 (New Englan d Biolabs) into the ScaI restriction site and thus tetrasa Ecrine resistance was imparted, but not ampicillin resistance. Got plus The mid was named pBN1.Construction of pBN4   The pA1 plasmid was opened at the HindIII and EcoRV sites. , Synthetic oligonucleotide 5'-A GCT GAA TTC AAC GTT   CTC GAG GAT-3 '(SEQ ID NO: 9) and its complementary sequence 5'-A TC CTC GAG AAC GTT GAA TTC-3 ′ (SEQ ID NO: 1 0) was cloned into the vector. By inserting these oligonucleotides, Unique EcoRI and XhoI restriction sites at the carboxyl terminus of hCAII The T7-hCAII cassette contained in was obtained. The resulting plasmid was named pA3 Was.   The pBN1 vector was digested with EcoRI, and the single-stranded overhang was Filled with ZeI large (Klenow) fragment. Straight with newly generated blunt ends The linear plasmid was religated, thus destroying the EcoRI site. The obtained plastic The sumid was named pBN3.   Plasmid pA3 was digested with restriction enzymes XbaI and BspEI. T7-h The DNA fragment from the pA3 digest containing the CAII cassette was converted to Xba Subcloned into pBN1 vector digested with I and BspEI. Obtained The resulting vector was named plasmid pBN4.Embodiment 2. FIG. Inoculation preparation of hCA fusion construct   121 ° C, 20 min in an autoclave in which L-broth is set in a liquid cycle Bacteria. Glucose and tetracycline stock solutions were added to a 0.22 μm filter. Filtered on a filter. Put the following solution into each of two 250 ml shake flasks Was:   50 ml L-broth (1.0% tryptone, 1.0% NaCl, 0.5% yeast extract)   1.0ml glucose stock solution (50mg / ml)   150 μl tetracycline stock solution (5.0 mg / ml)   Transfer with 100 μl of the vector encoding the desired hCA-fusion construct E. coli cell lysed inoculum   The shake flask was placed in a 37 ° C. incubator shaker (200-220 rpm, 1 0-14 hours). Then, the optical density (OD) of the cells in the resulting solution Was measured at 540 nm. Normally, a 1: 1.25 dilution is required to obtain a proper reading. I needed a pardon. Then, one of the two shake flasks is replaced with the next shake flask. Selected to inoculate sets of Scos. 3 500ml shake flasks that Each contained the following sterile solutions:   200 ml L-broth (1.0% tryptone, 1.0% NaCl, 0.5% ferment Mother extract)   4.0 ml glucose stock solution (50 mg / ml)   600 μl tetracycline stock solution (5.0 mg / ml)   1.0 ml inoculum from one of the first two shake flasks   The three shake flasks were placed on an incubator shaker under the above conditions and cells were Grow for -10 hours. Then, the optical density of the resulting solution was measured at 540 nm. (Typically 1: 1.25 dilution). Then, using all three shake flasks Fermenters were inoculated.Embodiment 3 FIG. Fermentative production of hCA-fusion constructs at 60L scale   The fermentation medium was placed in a fermenter, and the volume was adjusted to 45.0 L with distilled water. Culture The ground contained: 120.0 g casamino acid; 300.0 g yeast Kiss; 30.0 g NaCl; and 0.10 ml antifoam. Fermenta Was sterilized at 121 ° C. for 25 minutes. The fermenter was cooled to 37 ° C. Before inoculation Next, the following solution was added to the fermenter:   Glucose (800.0 ml H_Two480.0 g in O)   Magnesium (250.0 mlH_Two120.0 g MgSO in O_Four・ H_TwoO)   Phosphate (3.0 L H_Two120.0 g K in O_TwoHPO_FourAnd 465.0 g KH_Two PO_Four)   Tetracycline (30.0 ml 95% EtOH and 20.0 ml H_TwoIn O 0.90 g tetracycline / HCl)   The following mineral mixture (490.0 ml H_TwoDissolved in O and 10.0 ml concentrated hydrochloric acid ):         3.6g FeSO_Four・ 7H_TwoO         3.6 g CaCl_Two・ 2H_TwoO         0.90 g MnSO_Four         0.90 g AlCl_Three・ 6H_TwoO         0.09g CuCl_Two・ 2H_TwoO         0.18g molybdic acid         0.36g CoCl_Two・ 6H_TwoO   Except for the tetracycline and mineral mixture solution, all of the above solutions Sterilized in an autoclave with a body cycle for 20 minutes. Tetracycline and mi The neural mixture solution was sterilized by passing through a 0.22 μm filter. Typical At this point, the pH had dropped to about 6.5. If this happens, salt The pH was adjusted to 6.8 by addition of a group (14.8N aqueous ammonia). to pH 6.8 After reaching 600.0 ml of the inoculum was added to the fermenter. Time zero During the fermentation and during the fermentation, the following parameters were monitored: glucose concentration (about 2-5 g / L); optical density; pH (6.8 is near optimal); dissolved oxygen (40% is optimal). And the stirring speed. The temperature was maintained at 37 ° C. during the fermentation. Initial dissolved acid The element concentration was 90%, but dropped rapidly to 40%. Stirring speed and acid This level was maintained during the fermentation by increasing the elemental supplementation. Oxygen supplement Was started, the air inflow was reduced to 20 L / min. Initial glucose concentration The degree was about 9 g / L, but dropped to 5 g / L after about 6 hours. Glucose concentration If this drops to this level, the glucose feed (70% w / v glucose ) Was used to maintain the glucose concentration at 5 g / L.   When the fermentation has progressed to the point where the OD of 15-20 is measured, feed the medium. Started. The medium feed was 120.0 g potassium dissolved in 5.0 L of distilled water. Consists of zaminic acid and 300.0 g yeast extract, 20 minutes by liquid cycle It was sterile. The medium feed was fermented for 1.0-1.5 hours. Was added. When the fermentation progressed to OD 30.0, the following solution was Fermentation was induced by adding isopropylthiogalactoside ( IPTG; 28.8 g of 200 ml distilled water); ZnCl_Two(1 drop of 6N HCl (0.818 g) in 50 ml of distilled water containing Fill the IPTG solution with a 0.22 μm And sterilized with a filter. ZnCl_TwoUsing a liquid cycle autoclave For 20 minutes. After the addition, the IPTG concentration in the fermenter was 2.0 mM. Yes, ZnCl_TwoThe concentration was 100 μM. Next, the amino acid mixture fee Started. The amino acid feed was 225.0 g L-serine; 75.0 g L -Tyrosine; 74.0 g L-tryptophan; 75.0 g L-phenylalanine 75.0 g L-proline; and 75.0 g L-histidine; 1.5L H_TwoIt was dissolved in a mixture of O and 500 ml concentrated hydrochloric acid. Incube The fermentation broth was transferred to the harvest tank for about 2.0 hours, Cooled to 10 ° C. Typically, the fermentation yield is 6.5 as a wet cell paste 9.5 kg (dry cell weight about 1.0-1.5 kg).Embodiment 4. FIG. 60L fermentation harvest   Concentrate the cell suspension from the fermenter with a cross-flow membrane to reduce the volume. It was 10 L. The concentrated cell suspension was ultrafiltered and 30 L of cold wash buffer (50 mM Tris-SO_Four pH 7.8, 1.0 mM EDTA, and 0.10 mM Phenylmethylsulfonyl fluoride (containing PMSF). Then The cell suspension was concentrated to 8L. Typically for concentrating and washing cell suspensions Took 6-10 hours. Next, the concentrated cell suspension (cell paste) is packed in a bag. Frozen for later processing or homogenized with tank for cell lysis. Transferred to a nightizer.Embodiment 5 FIG. Cell lysis   The cell paste obtained from the 60 L fermenter is cooled with a washing buffer (above). , And the resulting cell suspension was cooled to 5-10 ° C. Cooling The cell suspension was homogenized at 12000 psi using a Galin high pressure homogenizer. I did Pass the homogenized cell paste through a heat exchanger to reduce the lysate to 10 ° C. Cooled and passed through a second homogenizer.Embodiment 6 FIG. Preparation of thrombin stock solution   Thrombin used for the fusion protein cleavage reaction was obtained from Calbiochem in lyophilized powder form. I got it. Concentration of 1 mg / ml when dissolved in Milli Q water and measured as specific activity The powder was solubilized before use.Embodiment 7 FIG. Production of PTH (1-34) (SEQ ID NO: 22)   hCAII, the interconnected peptide, and PTH (1-34) (SEQ ID NO: 22 Expression encoding a PTH construct comprising a DNA segment encoding By preparing the vector, a single copy of PTH (1-34) (SEQ ID NO: 22) A DNA segment encoding the fusion protein was prepared. Operon Tec The following oligos were obtained from hnologies Inc, 1000 Atlantic Ave. Alameda CA 94501.   Eight synthetic DNA oligos were obtained, the complementary sequences were phosphorylated and annealed. Ringed. Using four double-stranded fragments, PTH (1-34) (SEQ ID NO: No .: 22) encodes an interpeptide linker followed by DNA fragments were prepared. This DNA fragment is converted into the restriction site H digested with indIII and EcoRV, inserted into pA1, pA1: PTH (1-34) was prepared.   The expression cassette was constructed using the restriction endonucleases XbaI and BspEI. A1: A vector taken from PTH (1-34) and digested with the same restriction endonuclease And inserted into the vector pBN1. This final vector was constructed using pBN1: PTH (1-34). ).   Vector pBN1 containing DNA encoding hCA-PTH fusion protein : Preparation and cultivation of E. coli cells transformed with PTH (1-34) More dissolved. The hCA-PTH fusion protein is PTH (1-34) (SEQ ID NO: 2). 2) A thrombin cleavage site (Gly-Pro-Arg) adjacent to the N-terminus of the sequence Through an amino acid sequence corresponding to PTH (1-34) (SEQ ID NO: 22) HCAII. The hCA-PTH fusion protein is E. coli BL21 F^- ompTr_B ^-m_B ^-(DE3) expressed as inclusion bodies in cells.                               Purification of inclusion bodies   The cell lysate from the 60 L fermentation was continuously flowed at 400 ml / min. The mixture was centrifuged at 20000 g with a centrifuge. Pellets are roughly enclosed in 300-500g Included body. Crude hCA-PTH inclusions were dissolved in 2M citric acid at 60 mg solids / m suspension (4-8 liters) at 50% pulse rate and 70% power. Communication. Clarify solubilized inclusion bodies by centrifugation at 20,000 g It has become.   Addition of 10N NaOH until the pH is 4-5 gives the hC in the supernatant. The A-PTH fusion protein was precipitated. 2000 h of precipitated hCA-PTH fusion protein Collected by centrifugation at 0 g. Next, the precipitated hCA-PTH fusion protein was added to 4 h Washed with -8 liters of 5% acetic acid / 45% EtOH in Milli-Q water and precipitated The hCA-PTH fusion protein was collected again by centrifugation. Then, hCA-PTH Wash the fusion protein twice with 4-8 liters of 100 mM EDTA, then Wash once with 4-8 liters of Milli-Q water and, after each wash, centrifuge for protein collected.                    Thrombin cleavage of PTH (1-34)   200 ml of 50 mM NaOH and 0.25% N-lauroyl sarcosine Is added to the bottle containing the pellet of the hCA-PTH fusion protein. . Place the bottle in a 37 ° C water bath to warm the pellet (typically 2-5 minutes). Promotes solubilization of the kit. Homogenize material until all large chunks dissociate I do. Contains 50 mM NaOH and 0.25% N-lauroyl sarcosine Adjust the pH to 11.6 to 11.9 with the appropriate solution. Sonicate the resulting solution To dissolve all hCA-PTH fusion protein pellets. Typically, Sonication output 2, pulser on, 2 at 70% duty cycle Run for a minute (small batches take less time). Anti-absorbance at 280nm Determine the protein concentration of the reaction solution. Ideally, the concentration should be 6-7 mg / ml (When a 60 L fermentation product is adjusted to 8 to 10 L). The concentration is 9mg / ml Higher than 50 mM NaOH / 0.25% N-lauroyl sarcosine. Dilute to 6-7 mg / ml with solution.   Stir the protein solution vigorously, adjust the pH to 8-8.2 with 1 M Tris-HCl. Let If the solution is cloudy, filter through a glass fiber filter. Then, The solution is filtered through a 0.45 μm cellulose acetate membrane, and the absorbance at 280 nm is measured. This determines the protein concentration. Add solid NaCl to a final concentration of 250 mM I do. Add 1 mg / ml thrombin stock solution and add protein to enzyme ratio 5000: 1 w / w. The solution is then stirred on a 37 ° C. water bath plate. You. Buffer A (95% water, 5% acetonitrile (ACN), 0.1% Fluoroacetic acid (TFA) to buffer B (95% ACN, 5% water, 0.1 % TFA) by HPLC using a C8 column with a gradient elution to Monitor response. The reaction is stopped by adding PMSF. Typically, 46- The completion of the reaction is determined by the disappearance of the peak corresponding to the starting material at 48 minutes. PMS Dissolve F in 95% EtOH and add directly to the vigorously stirred reaction mixture You. By the thrombin cleavage, 5.1 g was obtained from 60 g of the hCA-PTH fusion protein. PTH (1-34) (SEQ ID NO: 22) is obtained.                Citric acid precipitation of thrombin cut PTH material   Cleavage of the obtained human carbonic anhydrase (hCA-PTH fusion protein) ) And the remaining unhydrolyzed fusion protein. Precipitate from solution by adding to 150 mM and adding the desired peptide (PTH 1-34) (SEQ ID NO: 22) in the solution. Precipitated material removed by centrifugation I do. The supernatant is filtered through a 0.45 μm filter and then frozen at -80 ° C Or desalting directly on a C8 column. Yield from peptide in this step Is 85%.   If desired, 10-70% buffer B (aqueous acetonitrile / trifluoro Acetic acid (ACN / TFA) gradient with a preparative C8 column. PTH (1-34) (SEQ ID NO: 22) may be purified by chromatography. . Here, buffer A is 0.1% TFA, 95% water, 5% acetonitrile. Yes, buffer B is 0.1% TFA, 5% water, 95% acetonitrile . Alternatively, preparative eluting first with a 10-70% buffer B gradient The PTH (1-34) (SEQ ID NO: 22) sample may be purified by a C8 column. . Here, buffer A contains 5 mM HCl, 95% water, 5% acetonitrile and And buffer B. PTH (1-34) (SEQ ID NO: 22) And desalted using Here, buffer A is 10 mM acetic acid, and buffer B is Is 10 mM acetic acid in 50% aqueous ethanol.Embodiment 8 FIG. Production of GLP (7-36) -NH ₂ (SEQ ID NO: 27)   Phosphorylate six synthetic DNA oligos (1-6) and anneal complementary strands Ringed. Oligopairs 1 and 2 were replaced with pUC19 (New England Biolabs. (Commercially available from Co., Ltd.) between HindIII and KpnI. Then, Rigopairs 3 and 4 were inserted between KpnI and EcoRI of this vector, A vector named pUC19: GLP (7-22) was obtained. Oligo pair 5 III and EcoRI, the vector PB5: GLP (21-34) A FA was obtained. pUC19: GLP (7-22) was deleted with NarI and EcoRI. And digest PB5: GLP (21-34) AFA with ClaI and EcoRI From PB5: GLP (21-34) AFA containing oligos 5 and 6 Ligation into the cut pUC19: GLP (7-22) The fragment containing oligopairs 5 and 6 was converted to pUC19: GLP (7-22) to obtain pUC19: GLP (7-34) AFA. pUC 19: GLP (7-34) AFA was digested with HindIII, and GLP1 (7-3 4) Peptide mutual phosphorus followed by -Ala-Phe-Ala (SEQ ID NO: 30) The fragment containing the sequence encoding the car was transferred to pA4 and the vector pA4 : GLP (7-34) AFA was obtained. The hCAII gene in pA4 is methionine. -240 had been mutated to change to cysteine. Next, the expression cassette Using the restriction endonucleases XbaI and BspEI to pA4: G Digest from LP (7-34) and digest with the same restriction endonuclease Was inserted into the obtained vector pBN1. The resulting vector was transformed into pBN6: GLP (7- 34) Named AFA.   hCA-GLP1 (7-34) -Ala-Phe-Ala (“hCA-GLP 1-AFA ") Vector pBN6 containing DNA encoding the fusion protein : E. coli cells transformed with GLP (7-34) AFA were prepared as described above. , Cultured and lysed. The hCA-GLP1-AFA fusion protein is GLP1 (7-3 4) Present adjacent to the N-terminus of -Ala-Phe-Ala (SEQ ID NO: 30) sequence GLP (7-34) -Ala-Phe-Ala (distribution) via a methionine residue HCAII linked to the amino acid sequence corresponding to SEQ ID NO: 30) Was. The hCA-GLP1-AFA fusion protein is E. coli BL21 F^-ompTr_B ^-m_B ^-(DE3 ) Was expressed as inclusion bodies.                               Purification of inclusion bodies   The cell lysate is centrifuged at a flow rate of 400 ml / min using a continuous flow rotor of 20,000 g. Separation was performed to obtain a pellet containing 300 to 500 g of the crude inclusion body. Crude hCA -Suspension of GLP1-AFA inclusion body at 25% solids concentration in lysis buffer (1.2-2.0 L), homogenize with Galin homogenizer at 13000 psi did. The homogenized inclusion bodies were centrifuged at 20,000 g and 100 mM acetic acid The suspension was resuspended in a sodium (pH 4.5) solution and subjected to a second homogenization. Next The homogenized inclusion bodies were then resuspended in distilled water. Homogenate the resulting suspension. Nize and collect the inclusion bodies by centrifugation. Dissolve purified inclusion bodies in 1.5 M citric acid Thaw and clarify by centrifugation. Add NaOH until the pH is 4-5 To precipitate the hCA-GLP1-AFA fusion protein and isolate by centrifugation. did.                       Cyanogen bromide cleavage of hCA-AFA   The hCA-GLP1-AFA inclusion body was dissolved in a 2.0 M citric acid solution to give about 35- The concentration was 45 mg / ml. The solution was adjusted to pH 1.0 using concentrated HCl and the total protein 0.2 g of cyanogen bromide (CNBr) was added per gram. Protein by HPLC The concentration was determined. Argon was bubbled through the solution before CNBr addition. Argo The cleavage reaction was allowed to proceed for 4-5 hours under an atmosphere of oxygen and then 2 g / g CNBr. .2 g of DL-methionine was added and then stirred for 30 minutes to obtain residual C. NBr was neutralized.           Purification of GLP1 (7-34) -AFA (SEQ ID NO: 30)   The solution obtained by the CNBr cleavage reaction was dissolved in 10% (w / v) sodium sulfate in water. All proteins and peptides were precipitated from solution by dilution 1: 1 with the liquid. Sample After centrifugation at 20,000 g for 15 minutes, the supernatant was decanted. Distilled water (2 00 ml) was added to each centrifuge vial. The resulting mixture is homogenized and Centrifuged at 0000 g for 15 minutes. The supernatant is decanted and the same volume of 50 % Acetonitrile (ACN) was added to the pellet material. Homogenize the sample The mixture was stirred for 10-15 minutes and finally centrifuged at 20000 g for 15 minutes. 50% Ase The extraction with a tonitrile solution was repeated two or three times to obtain GLP1 (7-34) A FA (SEQ ID NO: 30) was obtained with 90-95% recovery in acetonitrile solution. The extraction may be performed using 50% n-propanol.             Loading of 50% ACN extraction batch on DOWEX-1   DOWEX-1 (250 mg each of GLP1 (7-34) -AFA (SEQ ID NO: 30) 25 g) is pre-washed with 10% ACN before adding 50% ACN extraction solution . Load DOWEX-1 by batch method and stir for 20 minutes. Next, GLP1 (7 DOWEX-1 is filtered from the −34) -AFA (SEQ ID NO: 30) solution.                         Low pressure C8 chromatography   The 50% ACN solution was diluted with distilled water to 12.5% ACN and loaded onto C8 resin. Facilitates binding of GLP1 (7-34) -AFA (SEQ ID NO: 30). Pepti Load solution onto C8 resin in a low pressure column and 5 times column volume to 30% (V / v) Wash column with ACN solution. Then, GLP1 (7-34) -A The FA (SEQ ID NO: 30) is eluted with a 50% ACN solution in distilled water. 50% A The CN solution is freeze-dried to obtain GLP1 (7-34) -AFA (SEQ ID NO: 30). You. GLP1 (7-36) NH_TwoGLP1 (7-34) A into (SEQ ID NO: 27) Transpeptidation of FA (SEQ ID NO: 30)   GLP1 (7-34) AFA (SEQ ID NO: 30) (1 mmole) was added to 5 mM  CaCl_TwoAnd 400 mM Gly-Arg-NH_TwoDMF / H containing_TwoO In a 50/50 (v / v) solution and the pH of the solution was adjusted to 8. using 5M NaOH. Set to 0. Then 2 mg of TPCK-treated trypsin is added. GPL1 (7-36) NH_TwoTranspeptidation reaction resulting in (SEQ ID NO: 27) Is completed in 2-3 hours at room temperature.   If desired, aqueous acetonitrile / trifluoroacetic acid (ACN / TFA) By chromatography on a preparative C8 column using a fur gradient , GLP1 (7-36) -NH_Two(SEQ ID NO: 27) may be further purified.Embodiment 9 FIG. Production of GRF (1-44) NH ₂ (SEQ ID NO: 20)   Host eight oligonucleotides, including linker and peptide segments. Horizon, complementary oligonucleotide pairs 1 and 2, 3 and 4, 5 and 6, and 7 and 8 were annealed. Oligonucleotide pairs -1 and 2 and 3 and 4 were transformed with the pTZ19R vector (Pharmacia Biotec). h Inc., available from NJ) between the HindIII and SalI sites. And pTZ: GRF (1-29) was obtained. Oligonucleotide pair 5 And 6, and 7 and 8 were replaced with SalI and Eco of the pTZ19R vector. Simultaneous ligation between the RI sites yielded pTZ: GRF (29-44) A. pTZ: GRF (1-29) and pTZ: restrict GRF (29-44) A to endonuclease Digested with Xase I and SalI and pTZ: GRF (1-29) A 1.9 kb band was obtained from the vector and pTZ: GRF (29-44) A 0.9 kb band was obtained from the A vector and ligated, The child fragments are cloned into a single vector adjacent to each other and Z: GRF (1-44) A was obtained.   By site-directed mutagenesis for specific codons in plasmid pA1, h Three Asn-Gly sites in CAII (positions 10-11, 61-62 and 2 30-231), Gln10-Gly11, Gln61-Gly62 and And Asn230-Ala231. All three mutations are combined Sumid pA2 was created.   The pA2 vector was digested with EcoRV. pTZ: GRF (1-44) A to D digested with raI and EcoRV. Fragments containing GRF gene and direct The pA2 plasmid thus ligated was ligated to obtain pBN2: GRF (1-44) A. Was.   Vector pBN containing DNA encoding hCA-GRF fusion protein 2: E. coli cells transformed with GRF (1-44) A were prepared as described above and cultured. Nourished and dissolved. The hCA-GRF fusion protein binds to GRF ( HC bound to the amino acid sequence corresponding to 1-44) -Ala (SEQ ID NO: 31) AII. The interconnecting peptide is responsible for the thrombin cleavage site and It contained an enzyme cleavage site. Enterokinase cleavage site (Asp-Asp-A sp-Asp-Lys) (SEQ ID NO: 2) is GRF (1-44) -Ala (sequence No. 31) Present adjacent to the N-terminus of the sequence. HCA-G after thrombin treatment The RF fusion protein is the N-terminal of the GRF (1-44) -Ala (SEQ ID NO: 31) sequence. (Ala-Met-Val-Asp-Asp-Asp) -Asp-Asp-Lys) (SEQ ID NO: 19) Thrombin cleavage site (Gly-Pro-Arg) was present to produce . The hCA-GRF fusion protein is E. coli BL21 F^-ompTr_B ^-m_B ^-Inclusion body in (DE3) Was expressed as                               Purification of inclusion bodies   Cell lysate at 20,000 g in continuous flow rotor at 400 ml / min Centrifuged. The pellet contained 300-500 g of crude inclusions. Crude hCA -Suspend GRF-Ala inclusions in 2M citric acid at a concentration of 60 mg solids / ml. Cloudy (5-9 liters), sonicated at 50% pulse rate, 70% power . The solubilized inclusion bodies were clarified by centrifugation at 20,000 g.   Addition of 10N NaOH until the pH is 4-5 gives the hC in the supernatant. The A-GRF-Ala fusion protein was precipitated. Precipitated hCA-GRF-Ala fusion The combined proteins were collected by centrifugation at 20,000 g. Then, the precipitated hCA-GR The F-Ala fusion protein was washed with 5% acetic acid / 45% EtOH in Milli-Q water and precipitated. The hCA-GRF-Ala fusion protein was collected again by centrifugation. Then, The hCA-GRF-Ala fusion protein was resuspended twice in 100 mM EDTA. Then, the fusion protein was collected by centrifugation. Washed and precipitated hCA-GRF-A The la fusion protein was suspended in distilled water. The suspension was homogenized, and hCA-GRF- Ala fusion protein was collected by centrifugation.                   Thrombin cleavage of hCA-GRF fusion protein   Solution containing 50 mM NaOH and 0.25% N-lauroyl sarcosine 200 ml of the bottle containing the pellet of the hCA-GRF-Ala fusion protein. Was added. The bottle was placed in a 37 ° C. water bath to warm the pellet. NaOH solution Remove all solid material from the bottle wall by rotating the bottle along the wall of the bin did. The slurry was then poured into an appropriately sized beaker. Homogenize the material To dissociate all large lumps and monitor the pH. 50 mM NaOH PH of 11.6 with a solution of 0.25% N-lauroyl sarcosine. And adjusted to between 11.9. Sonicate the solution and pellet all inclusion bodies Was dissociated and the solution became clear. Sonication time depending on batch size changed. Typically, for 1 liter or more, 2 minutes, output 10, pulsar On, sonicated at 70% duty cycle (small batches require time Did not start).   The protein concentration of the reaction solution was measured. If the concentration is higher than 9 mg / ml, The solution was diluted with 50 mM NaOH / 0.25% N-lauroyl sarcosine solution and The white density was 6-7 mg / ml. The protein solution was stirred vigorously and 1M Tris- The pH was adjusted to 8-8.2 with HCl. At this time, if the solution is slightly turbid The solution was clarified by filtration through a glass fiber filter. Clarify The solution was sterile filtered through a 0.45 μm cellulose acetate membrane. At 280 nm The protein concentration was determined by measuring the absorbance. 250 mM NaCl concentration NaCl was added with vigorous stirring.   A thrombin stock solution sufficient to bring the ratio of protein to enzyme to 5000: 1 (1 mg / ml) and the resulting mixture is placed in a 37 ° C. water bath with a stirring plate. Stirred above. H_TwoC eluting with a gradient of O / ACN / TFA buffer The reaction was monitored by HPLC using 8 columns. The peak of the fusion construct is the book When qualitatively disappeared (typically 46-48 hours), PMSF was added to a final concentration of 0 The reaction was stopped by adjusting to 0.1 mM. Dissolve PMSF in 95% EtOH And added directly to the vigorously stirred reaction mixture.                     Citrate precipitation of hCA-fragment   By precipitation with 90 mM citric acid, the heat generated by cleavage of the fusion protein was Carbonic anhydrase fragment and the remaining uncleaved fusion protein The white was removed from the solution and the intermediate peptide Ldr-GRF (1-44) -Ala dissolved. It was left in the solution.   The volume of the thrombin cutting solution was measured with a graduated cylinder. 1M citric acid Stock solution was added to a final citric acid concentration of 90 mM. Stir solution vigorously While slowly adding. The flocculant was centrifuged and the supernatant was 0.45 μm Was filtered. Freeze the filtered supernatant at -80 ° C or desalinate Was directly loaded on a C8 column.   The pellet from the precipitation was washed with 90 mM citric acid (pH 3.0-3.1). Between 150 and 200 ml of citric acid dissolved in each pellet of 1-2 g of hCA The liquid was added. The pellet was homogenized, and the supernatant obtained by washing was washed with 0.45. The solution was filtered through a μm filter and stored. The washed pellet was discarded. Supernatant- Stored at 80 ° C. or desalted directly on a C8 column.               C8 desalting of Ldr-GRF (1-44) -Ala   Prepare a 90 mM citric acid solution of Ldr-GRF (1-44) -Ala Loaded directly on the ram. Column with aqueous ethanol / acetic acid buffer gradient Was eluted. Collect the Ldr-GRF (1-44) -Ala containing fractions (Reference (Typically 90-95% pure), the solution was concentrated by evaporation to 21-15 mg / ml And Store the resulting solution at -80 ° C until transpeptidation. Was.       Transpeptidation of Ldr-GRF (1-44) -Ala   The concentration of Ldr-GRF (1-44) -Ala peptide was adjusted to 1 mM GRF standard. Determined by C8 HPLC using the material. Dilute the peptide solution with water to 3 mM Ldr-GRF (1-44) -Ala. EDTA and sodium phosphate (5 mM; 1.9 mg / ml and 25 mM; 3.6 mg / ml, respectively). The pH was adjusted to 6.0 with 1 M NaOH. 2-nitrophenyl Lysinamide [ONGPA] (250 mM; 89.0 mg / ml) was added, and 1 The pH was adjusted again to 6.0 with M NaOH. Carboxypeptidase Y ("CP DY "; 2 µl / ml) and stirred at 35-40 ° C in the dark. Ldr The extent of the reaction producing -GRF-ONGPA was monitored by HPLC. About 1 After 2 hours, acetonitrile was added to a final concentration of 15% v / v. The reaction was stopped.   The sample obtained by CPD-Y transpeptidation was used for a C8 HPLC column. Load on top and buffer with 20% ethanol, 10 mM sodium phosphate, pH 6.8 The unreacted ONGPA was removed by washing with a filter. The column was then 50% ethanol , Elution was performed with a buffer of 10 mM sodium phosphate, pH 6.8, and Ldr-GRF- The peak portion of ONGPA was collected.                     Photolysis of Ldr-GRF-ONGPA   Ldr-ONGPA pair by C8 HPLC using 1 mM GRF standard. The peptide concentration was determined, diluted with 50% ethanol, and diluted with 1 mM Ldr-GRF-O. NGPA (6.0 mg / ml). Sodium bisulfite (5 mM) and And sodium benzoate (50 mM) and the solution was added with 1 M NaOH. The pH was adjusted to 9.5. After bubbling argon through the solution for 10-15 minutes, The mixture was irradiated while maintaining the temperature in a cyclic water bath at 20-25 ° C. (wavelength> 305 nm; 200-210 W medium pressure mercury lamp). During the reaction, maintain a constant flow of argon, The reaction was stirred at a constant speed. The reaction was monitored by HPLC. About 1-2 o'clock After a short time, the reaction was stopped by lowering the pH to 5.5 with acetic acid.       Ldr-GRF-NH_TwoEnterokinase Cleavage Protocol   Ldr-GRF-NH obtained by photolysis reaction_TwoSolution was diluted 1: 5 with water (1.0 mg / ml peptide). Add Triton X-100 to a final concentration of 0.1% did. Succinic acid and calcium chloride were added to a concentration of 50 mM (5 mM each). 0.9 mg / ml) and 2 mM (0.3 mg / ml) and dissolved in 10 M NaOH. The pH of the solution was adjusted to 5.5. After filtering the solution through a 0.45 μm membrane, 5.0 mg / Ml Dowex 1 resin was added. Add anion exchange Dowex 1 resin to the reaction Binding peptide containing Asp-Asp-Asp-Asp (SEQ ID NO: 32) sequence I let it. This reduces the required enterokinase concentration while improving substrate specificity As well as increase the reaction rate and net yield. 1 : Enterokinase enzyme in a ratio of 3000 (1 μl for 10 mg of peptide) Was added and the reaction was maintained in a 35-40 ° C. water bath with constant stirring. 2 After 0-24 hours, Ldr-GRF-NH2 was replaced with GRF (1-44) -NH2 (sequence The cleavage reaction to convert to No. 20) was 70-80% complete. Filter the reaction mixture To remove Dowex 1 and add acetonitrile to bring it back to 15% The reaction was further stopped. Samples were stored at -80 C until ready for purification. Desired Then, the product GRF (1-44)-was obtained by preparative HPLC using a C8 column. NH2 (SEQ ID NO: 20) may be purified.Embodiment 10 FIG. Production of peptides by expression of a soluble hCA fusion protein construct   Transformed with a vector containing DNA encoding the hCA-9AA fusion protein The obtained E. coli cells can be prepared and cultured by the above procedure. hCA-9A The A fusion protein has the amino acid sequence Thr-Asn-Thr via the interconnecting peptide. -Gly-Ser-Gly-Thr-Pro-Ala ("9AA") (SEQ ID NO: : 33) containing hCAII. The interconnected peptide is Enteroki Includes an enzyme cleavage site adjacent to the N-terminus of the 9AA (SEQ ID NO: 33) sequence In. The hCA-9AA fusion protein is E. coli BL21 F^-ompTr_B ^-m_B ^-At (DE3) And can be expressed as a soluble protein.   Dilute the cell paste from the fermenter with cold wash buffer, Cool down to The cooled cell suspension was removed using a Galin high pressure homogenizer for 1 hour. Homogenize at 2000 psi. Pass the cell paste through a heat exchanger to 10 ° C. And then perform a second homogenizer treatment. PM in 95% ethanol Add SF stock solution to a final PMSF concentration of 0.05 mM. Lysate about Cool to 8 ° C, pass through 12000 psi homogenizer, pool in storage tank I did. The lysate was passed through an additional 12000 psi homogenizer before the polyethylene Add a 5% v / v pH 7.5 solution of lenimide (PEI) to reduce the PEI concentration 0.35%. The resulting mixture is stirred for 20 minutes, 20000x C. Clarify with Westfalia. Pour the clarified solution into 0.22 μm Pall Filter through a filter system.   Affiliate containing p-aminomethylbenzene-sulfonamide-agarose resin Column with 0.1 M Tris-SO_FourEquilibrate with (pH 8.7) solution. P The supernatant clarified by the EI precipitation method was loaded on the column, and a series of Tris- SO_FourElute the column with the buffer solution. Column containing the fusion protein construct Pool traction. Use glacial acetic acid to lower the pH of the solution to 4.0. Causes the protein construct to precipitate from the pooled fractions. Precipitated fusion The protein construct can be isolated by centrifugation and lyophilized.   The precipitated fusion protein construct was purified using 50 mM Tris and 1 mM CaCl_TwoIncluding Dissolve in pH 8.0 buffer. Sonication of the mixture and everything Is dissolved. After the solution was filtered through a 0.45 μm membrane, 5.0 m g / ml Dowex 1 resin was added. Enterokinase enzyme in a ratio of 1: 3000 (1 μl per 10 mg of peptide) and stirring the reaction at a constant speed. Maintained in a 35-40 ° C water bath for 20-24 hours. Filter the reaction mixture to Dowex 1 The reaction is removed by adding acetonitrile to a final concentration of 15%. Stopped. 3: 1 volume of 15% (v / v) acetic acid was added to the solution and the resulting mixture Was stirred for 30 minutes, thereby precipitating the peptide fragment containing hCA. Let The hCA fragment was removed by centrifugation and 9AA (SEQ ID NO: 33) A supernatant containing the peptide is obtained. The supernatant is desalted on a C8 column and stored at -80 ° C. Good.   The invention has been described with reference to various specific and preferred embodiments and methods. However Many changes and modifications may be made without departing from the spirit and scope of the invention. Good will be apparent to those skilled in the art.   All publications and patent applications mentioned in this specification are to be of ordinary skill in the art to which this invention belongs. It indicates the technical level. All publications and patent applications are, by reference, To the extent that each publication and patent application is individually and individually indicated by reference As described herein.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 14/605 C07K 14/605 14/635 14/635 19/00 19/00 C12N 1/21 C12N 1/21 C12P 21/02 ZNA C12P 21/02 ZNAC // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB , GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), AL, AM, AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI , GB GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT , RO, RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, UG, UZ, VN (72) Inventor Henriksen, Dennis B. Lincoln, Nebraska, USA 68503 Street 343 Apartment 723 (72) Inventor Manning, Shane Di United States 68154 Nebraska Omaha, Seaward Plaza 11206 Apartment 2217 (72) Inventor De La Motte, Rebecca Es United States 68502 Nebraska Lin Khan, South Twenty-Fifth Street 1209 (72) Inventor Homequist, Burton United States of America 68526 Nebraska Lincoln, West Lake Shore Drive 442 No. (72) inventor Wagner, Fred W. United States 68,461 Nebraska Wo Ruton, box 77 Bee, route No. 1

Claims (1)

[Claims] 1. A method for producing a peptide, comprising precipitating a fusion protein construct comprising carbonic anhydrase and a variable fusion polypeptide. 2. The method of claim 1 wherein the fusion protein construct is precipitated from solution at a pH of about 3.2 to about 6.0. 3. 3. The method of claim 2, wherein precipitating the fusion protein construct further comprises adding a salt to the solution. 4. 4. The method of claim 3, wherein the salt comprises a divalent metal cation. 5. 3. The method of claim 2, wherein precipitating the fusion protein construct comprises adding an acid to the solution. 6. 3. The method of claim 2, wherein precipitating the fusion protein construct comprises adding a base to the solution. 7. The method of claim 1, wherein the fusion protein construct is precipitated from a solution containing ammonium sulfate or sodium sulfate. 8. The method of claim 1, wherein the fusion protein construct is precipitated from a solution containing a chaotropic agent. 9. 9. The method of claim 8, wherein the chaotropic agent comprises guanidine hydrochloride. 10. The method of claim 1, wherein the fusion protein construct is precipitated from a solution containing urea. 11. 2. The method of claim 1, wherein isolating the fusion protein construct comprises resolubilizing the precipitated fusion protein construct. 12. 12. The method of claim 11, wherein the precipitated fusion protein construct is resolubilized in a solution having a pH of at least about 10. 13. 12. The method of claim 11, wherein the precipitated fusion protein construct is resolubilized in a solution having a pH of less than about 3.2. 14． 12. The method of claim 11, wherein the fusion protein construct precipitated in a solution containing a chaotropic agent or a detergent is resolubilized. 15. The method of claim 1, further comprising cleaving the fusion protein construct to obtain a soluble carbonic anhydrase fragment and a soluble variable fusion polypeptide fragment. 16. The method of claim 15, further comprising precipitating a carbonic anhydrase fragment. 17． The method according to claim 15, further comprising contacting a solution of the carbonic anhydrase fragment with a support containing a benzenesulfonamide compound or an acetoazolamide compound. 18. The variable fusion polypeptide is selected from the group consisting of GRF (1-41) (SEQ ID NO: 23), GLP1 (7-34) (SEQ ID NO: 21), and PTH (1-34) (SEQ ID NO: 22). The method of claim 1, comprising an amino acid sequence corresponding to the selected peptide. 19. The method of claim 1, further comprising expressing the fusion protein construct in a host cell as part of an inclusion body. 20. 20. The method of claim 19, further comprising dissolving the inclusion body to obtain a solubilized fusion protein construct. 21. 21. The method of claim 20, wherein the inclusion body is dissolved in a solution containing citric acid. 22. 21. The method according to claim 20, wherein the inclusion body is dissolved in a solution containing a chaotropic agent or a surfactant. 23. 21. The method of claim 20, wherein the inclusion bodies are dissolved in a solution having a pH of at least about 10. 24. 2. The method of claim 1, wherein the variable fusion polypeptide comprises at least two copies of the target peptide. 25. The variable fusion polypeptide has the formula: -TargP- (CS2)-[-(Ln1) _n- (CS1) _m- TargP- (CS2)-] _r , where -CS1- and -CS2- are cleavage sites. ,-(Ln1)-is a linking sequence, -TargP- is a target peptide, n and m are 0 or 1, and r is an integer from 1 to about 150]. . 26. A peptide whose target peptide is selected from the group consisting of GRF (1-41) (SEQ ID NO: 23), GLP1 (7-34) (SEQ ID NO: 21) and PTH (1-34) (SEQ ID NO: 22) 26. The method of claim 25, comprising an amino acid sequence corresponding to 27. 2. The method of claim 1, wherein the carbonic anhydrase is linked to the variable fusion polypeptide by a cleavage site. 28. 28. The method of claim 27, wherein the cleavage site is a chemical cleavage site. 29. 28. The method of claim 27, wherein the cleavage site is an enzymatic cleavage site recognized by endopeptidase. 30. Cleaving a fusion protein construct comprising a carbonic anhydrase and a variable fusion polypeptide to obtain a soluble carbonic anhydrase fragment and a soluble variable fusion polypeptide fragment; and then precipitating the carbonic anhydrase fragment. A method for producing a peptide. 31. 31. The method of claim 30, further comprising extracting the precipitated carbonic anhydrase fragment with a solvent to recover a further amount of the variable fusion polypeptide fragment, wherein the solvent comprises an organic component. 32. 32. The method of claim 31, wherein the organic solvent comprises acetonitrile, propanol, citric acid, polyethylene glycol, or a mixture thereof. 33. 31. The method of claim 30, further comprising isolating the fusion protein construct. 34. 34. The method of claim 33, wherein isolating the fusion protein construct comprises precipitating the fusion protein construct. 35. 31. The method of claim 30, wherein the soluble carbonic anhydrase fragment is precipitated from solution at a pH of about 3.2 to about 6.0. 36. 36. The method of claim 35, wherein precipitating the soluble carbonic anhydrase fragment further comprises adding a salt to the solution. 37. 37. The method of claim 36, wherein the salt comprises a divalent metal ion. 38. 34. The method of claim 33, wherein isolating the fusion protein construct comprises contacting a solution of the fusion protein construct with a support comprising a benzenesulfonamide compound or an acetoazolamide compound. 39. 39. The method of claim 38, wherein contacting the fusion protein construct solution with the support comprises passing the fusion protein construct solution through an affinity column containing a sulfanilamide compound. 40. 34. The method of claim 33, further comprising expressing the fusion protein construct in a host cell as part of an inclusion body. 41. 34. The method of claim 33, wherein isolating the fusion protein construct comprises adding PEI to a solution of the fusion protein construct. 42. 42. The method of claim 41, further comprising adjusting the pH of the PEI solution to between about 3.2 and about 6.0, thereby precipitating the fusion protein construct. 43. A carbonic anhydrase is linked to the variable fusion polypeptide via an enzymatic cleavage site; cleaving the fusion protein construct can include enterokinase, factor Xa, ubiquitin cleaving enzyme, thrombin, trypsin, renin, subtilisin, 31. The method of claim 30, comprising treating the fusion protein construct with an endopeptidase selected from the group consisting of chymotrypsin, clostripain, and S. aureus V8. 44. 44. The method of claim 43, wherein cleaving the fusion protein construct comprises treating the fusion protein construct dissolved in a solution containing sodium chloride with thrombin. 45. 44. The method of claim 43, wherein cleaving the fusion protein construct comprises adding an anion exchange resin to a solution of the fusion protein construct, and then treating the fusion protein construct with enterokinase. 46. A carbonic anhydrase is linked to the variable fusion polypeptide via a chemical cleavage site; cleaving the fusion protein construct comprises cyanogen bromide, hydroxylamine, BNPS-skatole, S-cyanylating agent and about the method of claim 30 in which comprises treating the fusion protein construct by chemical cleaving agents are selected from the group consisting of acids having less than 3.0 pK _a. 47. An inclusion body expressed in an E. coli host cell comprising a fusion protein construct, wherein the fusion protein construct comprises a carbonic anhydrase and a target peptide, wherein the target peptide is a GRF (1-41). ) (SEQ ID NO: 23), GLP1 (7-34) (SEQ ID NO: 21) and PTH (1-34) (SEQ ID NO: 22). An inclusion body that is a waste. 48. 48. The inclusion body of claim 47, wherein the fusion protein construct comprises at least two copies of the target peptide. 49. The target peptide comprises an amino acid sequence corresponding to PTH (1-34) (SEQ ID NO: 22); and the carbonic anhydrase is linked to the target peptide via an interconnected peptide containing an enzymatic cleavage site recognized by thrombin. 48. The enclosure of claim 47, wherein the enclosure is connected to. 50. The target peptide comprises an amino acid sequence corresponding to GRF (1-41) (SEQ ID NO: 23); and the fusion protein construct is recognized by an enzyme selected from the group consisting of thrombin, enterokinase and carboxypeptidase Y. 48. The inclusion body of claim 47, comprising an enzymatic cleavage site. 51. 49. The inclusion body of claim 48, wherein the target peptide comprises an amino acid corresponding to GRF (1-44) (SEQ ID NO: 20). 52. 48. The target peptide comprises an amino acid sequence corresponding to GLP1 (7-34) (SEQ ID NO: 21); and the fusion protein construct further comprises an enzymatic cleavage site recognized by trypsin. Inclusions. 53. 48. The inclusion body of claim 47, wherein the target peptide comprises an amino acid sequence corresponding to GLP1 (7-34) (SEQ ID NO: 21); and wherein the fusion protein construct comprises a methionine residue. 54. Expressing the fusion protein construct in an E. coli host cell as part of an inclusion body, wherein the fusion protein construct comprises carbonic anhydrase and a variable fusion polypeptide; then isolating the fusion protein construct A method for producing a peptide, comprising: 55. The variable fusion polypeptide is selected from the group consisting of GRF (1-41) (SEQ ID NO: 23), GLP1 (7-34) (SEQ ID NO: 21) and PTH (1-34) (SEQ ID NO: 22) 55. The method of claim 54, comprising a target peptide comprising an amino acid sequence corresponding to the peptide to be made. 56. 55. The method of claim 54, wherein the variable fusion polypeptide comprises at least two copies of the target peptide. 57. A fusion protein construct comprising a carbonic anhydrase and a variable fusion polypeptide, wherein the variable fusion polypeptide comprises a target peptide and the target peptide is GRF (1-41) (SEQ ID NO: 23) , A fusion protein comprising an amino acid sequence corresponding to a peptide selected from the group consisting of GLP1 (7-34) (SEQ ID NO: 21) and PTH (1-34) (SEQ ID NO: 22). 58. The variable fusion polypeptides are GRF (1-41) (SEQ ID NO: 23), GRF (1-44) (SEQ ID NO: 20), GLP1 (1-34) (SEQ ID NO: 26), GLP1 (7 -34) (SEQ ID NO: 21), GLP1 (7-36) (SEQ ID NO: 27), GLP1 (7-37) (SEQ ID NO: 28), PTH (1-34) (SEQ ID NO: 22), PTH 58. The fusion protein construct of claim 57, comprising a target peptide corresponding to a peptide selected from the group consisting of (1-38) (SEQ ID NO: 29) and PTH (1-84) (SEQ ID NO: 25). 59. 58. A recombinant gene comprising a DNA sequence encoding the fusion protein construct of claim 57. 60. 58. An expression cassette comprising a nucleic acid sequence encoding the fusion protein construct of claim 57, wherein the nucleic acid sequence is operably linked to a promoter functional sequence in a vector. 61. An expression vector comprising the expression cassette of claim 60. 62. 58. A transformed cell comprising a recombinant gene comprising a DNA sequence encoding the fusion protein construct of claim 57. 63. Cleaving the fusion protein construct comprising the carbonic anhydrase and the variable fusion polypeptide to obtain a carbonic anhydrase fragment and a variable fusion polypeptide fragment; precipitating the carbonic anhydrase fragment and the variable fusion polypeptide fragment A method for producing a peptide, comprising extracting the precipitated fragment with a solvent containing an organic component, thereby recovering the variable fusion polypeptide fragment. 64. 64. The method of claim 63, wherein the organic solvent comprises acetonitotyl, propanol, citric acid, polyethylene glycol, or a mixture thereof. 65. 64. The method of claim 63, wherein the variable fusion polypeptide comprises at least two copies of the target peptide.