WO2004044007A1

WO2004044007A1 - Production of ubiquitin fusion proteins

Info

Publication number: WO2004044007A1
Application number: PCT/AU2003/001491
Authority: WO
Inventors: Stan Bastiras; Angelo Guidolin; Chris Giannakis
Original assignee: Bresagen Limited
Priority date: 2002-11-12
Filing date: 2003-11-12
Publication date: 2004-05-27
Also published as: WO2004044007A8

Abstract

Methods for the production of protein or peptides are described. The method generally comprises at least the steps of: a) producing a fusion protein/peptide comprising Ubiquitin fused to the N terminus of a protein/peptide of interest, and a leader sequence (L), said leader sequence being fused to the N-terminus of the. Ubi; b) conducting a cleavage reaction in which said fusion protein is contacted with a ubiquitin protease (UbP) to at least partially cleave the ubiquitin from the protein or peptide of interest; c) contacting the products of said cleavage reaction with an ion exchange column; d) selectively eluting the protein or peptide of interest from the column; and e) capturing the protein or peptide of interest.

Description

PRODUCTION OF UBIQUITIN FUSION PROTEINS

FIELD

The present invention relates to methods for the production of proteins or peptides of interest. Particularly the invention relates to novel leader-ubiquitin fusion proteins and the production of protein or peptides of interest by expression of ubiquitin fusion proteins in bacteria.

BACKGROUND There is generally a widespread demand for proteins with commercial and clinical applications, and large scale, efficient and cost-effective processes are required for their production. Small peptides may be produced efficiently by chemical synthesis, but this process becomes more arduous and inefficient with larger peptides. Accordingly gene expression may be the preferred method for production for proteins and larger peptides.

In one approach the protein or peptide of interest is synthesised as a ubiquitin- fusion (Ubi- fusion) protein by expression of a gene construct in bacteria. The Ubi-fusion protein may be produced in a soluble form (for example, secreted into the periplasmic space), or alternatively as insoluble inclusion bodies (IBs).

The ubiquitin-fusion protein is then cleaved with ubiquitin protease (UbP) to form ubiquitin and the natural protein or peptide of interest. Cleavage of the Ubi-fusion protein maybe conducted in vivo or in vitro. Cleavage in vivo is achieved by coexpression of UbP and occurs in close association with translation of the Ubi-fusion protein. Cleavage in vitro is conducted after isolation of the Ubi-fusion protein from the host bacteria.

UbP Ubi-fusion protein ► Natural protein + Ubiquitin Efficient production of some Ubi-fusion proteins in bacteria has been difficult to achieve. For example poor yields of Ubi-fusion proteins containing IGF1 and E21R (a GM-CSF antagonist closely related to GM-CSF in structure) are routinely obtained by bacterial expression. These poor yields may be associated with inefficient expression and/or protein instability. Surprisingly the yields of Ubi-fusion proteins, including Ubi-IGFl and Ubi- E21R was increased significantly when a leader sequence was added to the N-terminal end of Ubi-fusion proteins. Improvement in yields may be achieved with Ubi-fusion proteins produced in a soluble form, or as insoluble IBs.

Most clinical and commercial appUcations of protein or peptides of interest require that they are in essentially pure form, essentially free of ubiquitin and uncleaved Ubi-fusion protein.

Accordingly an efficient purification process is required to remove Ubi and uncleaved Ubi- fusion protein from the protein or peptide of interest. Generally, it is preferable to include an ion exchange chromatography step in the purification process, since it involves the use of aqueous solvents, and allows simple, large scale enrichment with relatively low associated costs. Other purification steps may include hydrophobic interaction chromatography, reverse phase chromatography and/or additional ion exchange chromatography.

There is considered to be widespread difficulty in using ion exchange chromatography to separate a protein or peptide of interest from its uncleaved Ubi-fusion protein. The difficulty arises because the addition of Ubi to the N-terminus of a required protein or peptide generally does not alter the pi significantly. This near identity in the pi of required protein or peptides and their Ubi-fusion proteins is apparent at least for a large number of proteins; Table 1 provides examples thereof. The consequence of this similarity in pi is that the ion exchange elution profiles of the desired protein and the Ubi-fusion protein are closely aligned, thereby hindering efficient separation. Purification of the native protein by ion exchange chromatography is facilitated by addition of a N-terminal leader sequence containing one or more charged residues to the Ubi-fusion protein. The charged leader sequence creates a pi difference between the uncleaved [leader-Ubi-fusion protein] and the cleaved native protein, resulting in distinctly different elution profiles. The addition of a leader sequence to a Ubi-fusion protein for the purpose of facilitating purification of the protein of interest is appUcable to Ubi-fusion proteins and proteins or peptides of interest produced either as insoluble IBs, or in a soluble form.

Table 1

Effect on the Isoelectric Point (pi) of a Protein/peptide by the Addition of Ubiquitin to its N-terminus

Protein/peptide pi* pi of ubi-X fusion*

human IL-2 7.16 7.22 human thrombopoeitin 9.52 9.36 human erythropoeitin 8.09 8.08 human myoglobin 7.61 7.63 bovine ribonuclease 8.24 8.25 human interferon receptor G-28 8.19 8.18 chicken lysozyme 9.11 9.07 human interferon receptor-αl 6.51 6.63 human growth hormone 5.14 5.42 human GM-CSF(E21R) 5.53 5.82

*data were obtained by inserting amino acid sequence directly into DNAsis isoelectric point calculation algorithm

Although proteins may be produced in bacteria in a soluble form, or alternatively as insoluble IBs , there are major advantages in producing proteins in bacteria as insoluble

IBs, related to ease and simplicity of purification. For example isolation of inclusion bodies from bacteria by centrifugation, the first step in purification, separates the desired protein from greater than 90% of contaminating bacterial debris, including nucleic acids, lipids and bacterial proteins. Redissolved inclusion bodies are then refolded and the desired protein can be purified by conventional chromatography procedures. The relative simplicity of the inclusion body approach also has associated low cost advantages that are important for commercial scale protein production.

Inclusion body formation is closely related to the size and amino acid sequence of the protein, and there are difficulties in forming inclusion bodies with peptides and small proteins. For example ubiquitin itself (which has 76 amino acids) does not readily form inclusion bodies, and similarly robust inclusion body formation does not occur with small ubi-fiision proteins. The inventors have found that inclusion body formation with small ubi-fusion proteins is improved significantly when an N-terminal leader sequence is added to the N-terminus of a ubi-fusion protein, thus allowing preparation of native small proteins using the simple, low cost procedures associated with inclusion body purification procedures.

STATEMENT OF INVENTION

The present invention generally relates to improvements in the production of protein or peptides via the commonly utilised ubiquitin-fusion protein method, as outlined in the "Background" section hereof. The invention is realised by using a Ubi-fusion protein in which a leader sequence is fused to the N-terminus thereof.

In one broad aspect of the present invention there is provided a fusion protein comprising Ubiquitin (Ubi) fused to the N-terminus of a protein or peptide of interest, and a leader sequence (L) fused to the N-terminus of Ubi. The leader sequence ( ) may include one or more amino acids. Preferably the leader sequence includes between 5-100 amino acids. Preferably the leader sequence includes between 5 - 100 amino acids selected from the group listed in Table 2. Table 2

Amino acids single letter code

Alanine A

Arginine R

Asparagine N

Aspartic acid D

Cysteine C

Glutamic acid E

Glutamine Q

Glycine G

Histidine H

Isoleucine I

Leucine L

Lysine K

Methionine M

Phenylalanine F

Proline P

Serine S

Threonine T

Tryptophan W

Tyrosine Y

Valine V

In respect of the purification of a protein or peptide of interest, the inventors have identified that the addition of a charged leader sequence to the N-terminus of a Ubi-fusion protein allows the use of ion exchange chromatography to achieve efficient separation of the protein or peptide of interest from contaminating Ubi and/or uncleaved Ubi-fusion protein.

In one embodiment the leader sequence provides a locally charged region of the Leader- Ubi-fusion protein (L-Ubi-fusion protein) molecule that is able to interact strongly with the ion exchange support. The inventors have observed efficient separation utihsing such a leader sequence even when the overall pi of the L-Ubi-fusion protein may not differ significantly from the pi of the leaderless Ubi-fusion protein or the protein or peptide of interest.

In one embodiment of the invention the leader sequence contains one or more charged residues.

More preferably the leader sequence contains two or more charged residues. Preferably where two or more charged residues are present within the leader sequence each residue has an equivalent charge; ie either all charged residues are positive, or all charged residues are negative. It will be appreciated that the overall electrical charge of the leader sequence may be altered depending on, for example, whether anion exchange or cation exchange is to be used to separate the protein or peptide of interest. "Charged residues" are taken to include naturally occuring amino acids Asp, Glu, Lys and Arg. Those of general skill in the art to which the invention relates will readily appreciate unnatural amino acids and/or derivatives thereof, or modified amino acids that may be applicable to the invention.

Preferably that the leader sequence include the acidic residues, Asp and/or Glu where anion exchange chromatography is desired to be used in the purification process. In the case of cation exchange chromatography-based separation, the leader sequence should preferably include the basic residues Arg and/or Lys.

Preferably the leader sequence is chosen from the group consisting: MFATSSSTGNDG; MFATSSSTGDDG;

MFATSSSDGDDG;

MFATSDSDGDDG;

MFATSSDTDNDG;

MFATDSDTDNDG;

MFADNDG;

MFATSSSTGND;

MPATSSSTGNTMKEVKSLLLEFI;

MAPTSSSTGNTlV_K_BVKSLLLDLQLLLEKVKNPENR_ .SRMHTFEFI;

MKEN SLLLEFI;

MKENDSLLLEFI;

M ENKSLDLEFI;

MKENDSLDLEFI;

MKE tKSLLLDLQLLLEKV__ PEΝRKLSRMHTFEFI; and

M_-ENKSLLLDLQLLLEKVKNPENRKLSRMHTFDFYVPKNNATEFI.

Preferred charged leader sequences include

MFATSSSTGNDG;

MFATSSSTGDDG;

MFATSSSDGDDG;

MFATSDSDGDDG;

MFATSSDTDNDG;

MFATDSDTDNDG; and

MFADNDG.

Leader sequences are given in single letter amino acid code. The optimum number of charged residues within the leader sequence may readily be determined by a skilled person through routine testing.

The protein or peptide of interest may be any protein or peptide which has at least 5 amino acids, preferably at least 10 amino acids, and may include hGH, E21R, Glucagon, IGF-1 , Relaxin, Parathyroid hormone (PTH) and parathyroid hormone fragments, Amyloid B- protein fragments, leptin, Leptin fragments (eg 22-56), B-endorphin fragment, Galanin, Somatostatin, Brain natriuretic peptide, Endothelin 2, Endothelin 1, Inhibin-like peptide, HIN envelope fragments,. Conotoxins, Antibodies, antibody fragments and Factor Xllla fragments.

One protein of interest is human growth hormone (hGH).

Alternatively the protein of interest is E21 R or IGF- 1.

Alternatively the protein of interest is PTH or frage ents thereof.. PTH(l-34) is particularly suited to the invention.

In a further broad aspect of the present invention there is provided a method for the improved production of a protein or peptide of interest in the form of a Ubi-fusion protein expressed in a recombinant cell wherein a leader sequence is fused to the Ν terminus thereof.

In a further broad aspect of the present invention there is provided a method for the improved production of a protein or peptide of interest in the form of a Ubi-fusion protein expressed in a recombinant cell wherein leader sequence is fused to the Ν-terminus thereof where said method includes at least the steps of: a) producing in a host cell a fusion protein comprising Ubiquitin fused to the N-terminus of a protein or peptide of interest, and a leader sequence (L), said leader sequence being fused to the N-terminus of the Ubi; b) conducting a cleavage reaction in which said fusion protein is contacted with a ubiquitin protease (UbP) to at least partially cleave the ubiquitin from the protein of interest; c) purifying the protein of interest from the products of said cleavage reaction.

Preferably said host cell is prokaryotic cell, most preferably a bacterial cell.

The Ubi-fusion protein may be produced as a soluble protein or produced as insoluble inclusion bodies. A soluble L-Ubi-fusion protein generally occurs in refolded form in vivo. The soluble L-Ubi-fusion protein may be cleaved in vivo or isolated from the host cell and cleaved in vitro. The soluble protein or peptide of interest is then purified by standard methods.

In an alternative method, the L-Ubi-fusion protein is cleaved in vivo, and the protein of interest is produced as insoluble IBs. In a preferred method, the L-Ubi- fusion protein is produced as inclusion bodies, the protein is redissolved and refolded by standard methods prior to cleavage in vitro with UbP.

The leader sequence may be any leader sequence, including those herein before described.

Preferably the leader sequence is a charged leader sequence.

The protein or peptide of interest may be any protein or peptide of interest, including those herein before described.

Preferably the protein of interest is human growth hormone (hGH). Alternatively the protein of interest is E21R or IGF1.

Alternatively the protein of interest is PTH(l-34).

In a further broad aspect of the present invention there is provided a use of a leader sequence fused to the N-terminus of a Ubi-fusion protein for the production of a protein or peptide of interest, wherein said leader sequence improves the production levels of the Ubi-fusion protein or peptide following expression thereof from a protein or peptide expression construct in a host cell.

In a further broad aspect of the present invention there is provided a use of a leader sequence fused to the N-terminus of a Ubi-fusion protein for the production of a protein of interest, wherein said leader sequence improves the production levels of the Ubi-fusion protein following expression thereof from a protein expression construct in a host cell.

Preferably said host cell is a prokaryotic cell, preferably a bacterial cell. Most preferably the bacterial cell is an E. coli strain such as E. coli K12 or E. coli B.

In another aspect of the invention there is a method for producing peptides or small proteins of interest, where the protein or peptide is composed of 10 or more amino acids where said method includes at least the steps of:

a) producing a fusion protein comprising Ubiquitin fused to the N-terminus of a protein of interest, and a leader sequence (L), said leader sequence being fused to the N-terminus of the Ubi, and where said leader causes the leader-

Ubi-fusion protein to be produced as insoluble inclusion bodies. b) Isolating the inclusion bodies from the bacteria, redissolving the inclusion bodies and refolding the leader-Ubi-fusion protein using standard refolding methods. c) conducting a cleavage reaction in which said fusion protein is contacted with a ubiquitin protease (UbP) to at least partially cleave the Leader- ubiquitin from the protein or peptide of interest; d) purifying the smaU protein or peptide of interest from the products of said cleavage reaction.

The leader sequence may be any of those herein before described.

The small protein peptide of interest may include somatostatin, glucagon, or a protein from the conotoxin family.

In a further aspect of the present invention there is provided a use of a leader sequence • fused to the N-terminus of a Ubi-fusion protein for the production of a small protein of interest, wherein said leader sequence causes the the leader-Ubi-fusion protein to be produced as insoluble inclusion bodies.

The preferred leader sequence to form insoluble inclusion bodies include,

MPATSSSTGNTMKEVKSLLLEFI; IVIAPTSSSTGNTMKEVl^LLLDLQLLLEKVK PENRKLSRMHTFEFI;

MKENKSLLLEFI;

MKEVDSLLLEFI;

MKEVKSLDLEFI;

MKEVDSLDLEFI; MKEλ^SLLLDLQLLLEKNKΝPEΝRKLSRMHTFEFI; and

1VIKENKSLLLDLQLLLEKNI ΝPEΝRKLSRMHTFDFYNPKVΝATEFI.

In another broad aspect of the present invention there is provided a method of purifying a protein or peptide of interest comprising at least the steps of: a) producing a fusion protein comprising Ubiquitin fused to the N-terminus of a protein or peptide of interest, and a leader sequence (L), said leader sequence being fused to the N-terminus of the Ubi, and containing one or more charged residues; b) conducting a cleavage reaction in which said fusion protein/peptide is contacted with a ubiquitin protease (UbP) to at least partially cleave the Leader-ubiquitin from the protein/peptide of interest; c) contacting the products of said cleavage reaction with an ion exchange column; d) selectively eluting the protein or peptide of interest from the column; and e) capturing the protein or peptide of interest.

Preferably the pH at cleavage is from 6.2 to 8.0. More preferably the pH at cleavage is about 7.4.

Preferably the pi of the cleaved leader-ubiquitin molecule is sufficiently different to the pi of the protein or peptide of interest to allow efficient separation of the leader-ubiquitin molecule from the protein or peptide of interest.

Preferably, where the leader sequence contains one or more of the charged residues .Asp and/or Glu the ion exchange column is an anion exchange column.

Alternatively, where the leader sequence contains one or more of the charged residues Arg and/or Lys the ion exchange column is a cation exchange column.

Preferably said leader sequence is chosen from the group herein before described.

Preferably the ion exchange column is an anion exchange column. In a further broad aspect of the present invention there is provided a method of producing hGH according to the methods herein described.

In a further broad aspect of the present invention there is provided a method of producing E21R according to the methods herein described.

In a further broad aspect of the present invention there is provided a method of producing IGF1 according to the methods herein described.

In a further broad aspect of the present invention there is provided a method of producing PTH(l-34) according to the methods herein described.

In another broad aspect of the invention there is provided the use of a charged leader sequence fused to the N-terminus of a Ubi-fusion protein to promote the separation of uncleaved Ubi-fusion protein from a protein or peptide of interest during ion exchange chromatography.

The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of 2 or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

FIGURES

These and other aspects of the present invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying figures, in which:

Figure 1 Illustrates elution profiles of products following cleavage of Ubi-hGH,

L(D)-Ubi-hGH and L(DD)-Ubi-hGH with UbP. The percentages refer to the amount of buffer B, a buffer containing 0.5M NaCl, when mixed in a gradient with buffer A, a buffer containing no salt, required to displace the protein peptide of interest from the ion-exchange support.

Figure 2 Illustrates (A) Fractionation of ubiquitin and various N-terminal Leader- ubiquitin fusion proteins. (B) Fractionation of various ubiquitin C-terminal glucagon (GGN) fusion proteins. (C) Fractionation of various ubiquitin C- teirninal IGF-1 fusion proteins. Arrows indicate the position of relevant protein species. Fractionation of ubiquitin derivatives were expressed in E. coli K12. Whole cell lysates (wcl) were separated into soluble, supernatant

(s) and insoluble, pellet (p) fractions. Samples were electrophersised on 4- 12% NuPAGΕ (No vex) and then stained with Coomassie Brilliant Blue.

PREFERRED ΕMBODIMΕNT(S)

The following is a description of the preferred forms of the present invention given in general terms. The invention will be further elucidated from the Examples provided hereafter.

As used herein the term "residue(s)" refers to amino acid(s). The term should be taken to encompass amino acids which occur naturally, in proteins or peptides or otherwise, unnatural amino acids and/or their derivatives, or amino acids to which side chain modifications may have been made (modified amino acids). Natural and unnatural amino acids and/or derivatives thereof, or modified amino acids which are applicable to the present invention will be readily recognised by those of skill in the art to which the invention relates.

Naturally occurring amino acids may be referred to herein using the standard one or three letter nomenclature commonly used in the art. Such nomenclature is elucidated in "Biochemistry", CK Mathews and KE van Holde, The Benjamin/Ciin-r ings PubUshing Company, Inc., 1990, for example. "A protein or peptide of interest" may be any protein or peptide which one has an interest in producing on any scale, including proteins or peptides which may be destined for medicinal or therapeutic applications. Proteins found to be of particular interest to the inventors of the present invention include human growth hormone (hGH), E21R and IGF 1.

The method of the present invention is referred to herein as providing a protein or peptide of interest in an "essentially pure form". This term should be taken to mean that the protein or peptide of interest is retrieved or captured following purification according to the method, in a form substantially free from ubiquitin and uncleaved L-Ubi-fusion protein . "Substantially free" from ubiquitin and uncleaved L-Ubi-fusion protein preferably refers to levels of L-Ubi tod uncleaved L-Ubi-fusion protein peptide of approximately less than 100 ppm. While not preferable, a protein or peptide of interest in an essentially pure form may contain other contaminating substances, however it will be appreciated that these should not exceed a level thought to be detrimental to the use to which the protein or peptide may subsequently be put.

The invention is appUcable to L-Ubi-fusion proteins expressed by any recombinant cells, particularly prokaryotic cells and more preferably bacterial cells.

Those of skill in the art will appreciate the overall charge of the leader sequences exemplified above and accordingly to what end they may be used in the invention.

The length of a leader sequence according to the invention may vary depending on a number of factors such as the size and nature of the protein or peptide to which it is fused (via ubiquitin), or stability of the protein or peptide during expression thereof. However, it will be appreciated that the leader sequence must be such that it does not hinder the correct folding of the Ubi-fusion protein/peptide, or sterically block the ubiquitin moeity necessary for recognition by a ubiquitin protease, both of which are thought to be required for correct cleavage of ubiquitin from the protein or peptide of interest. A Ubi-fusion protein having fused thereto a leader sequence according to the present invention may be produced by any means commonly used in the art to produce fusion proteins. For example, a suitable nucleic acid vector capable of expressing a fusion protein in a desired host cell may be constructed using standard recombinant techniques, subsequently transfected into the desired host by known means, and allowed to express the fusion protein (whether in an induced or constitutive manner). As previously discussed herein, once expressed, a fusion protein may be collected, for example, as insoluble inclusion bodies. In a preferred form where the L-Ubi-fusion protein/peptide is captured as inclusion bodies, standard in vitro refolding procedures may be employed. The protein or peptide of interest may then be liberated from its fusion partners by subjecting the L-Ubi- fusion protein/peptide to a standard in vitro cleavage reaction involving a ubiquitin protease. Alternatively the L-Ubi-fusion protein may be expressed as a soluble refolded protein, isolated from the host cell, and cleaved in vitro with a UbP . In yet another alternative, cleavage of the L-Ubi-fusion protein is conducted in vivo by co-expression with a UbP. Cleavage in vivo is closely associated with translation, and the protein or peptide of interest may occur in a soluble form, or the protein of interest may form insoluble IBs. It will be appreciated that the products of the L-Ubi-fusion cleavage reaction will generally include: uncleaved L-Ubi-fusion protein; L-Ubi; and, protein or peptide of interest.

Following the cleavage reaction a protein or peptide of interest may be purified or substantially separated from contaminating uncleaved L-Ubi-fusion protein or peptide and L-Ubi by subjecting the products of the cleavage reaction to ion exchange chromatography. It will be appreciated that either an anion exchange column or a cation exchange column may be utilised depending on the nature of the leader sequence employed (ie the electrical charge thereof), and the nature of the protein/peptide of interest, for example.

With respect to PTH(l-34) the inclusion bodies (IBs) containing the PTH fusion protein (FP) can be dissolved using a variety of methods such as low pH (15 mM HC1), high pH (15 mM NaOH) and combinations of high pH and chaotropic agents such as urea and/or guanidine hydrochlori.de. For example, IBs may be solubilised in 15mM HC1 or 15 mM NaOH at 0.1 to 10 mg/mL FP for periods up to lhr. Once dissolved, the protein is ready for refolding. If low pH dissolution has been used, refolding is initiated by adjusting the pH to 2-3.5, using NaOH. Alternatively, if high pH dissolution has been used, the pH is lowered to pH 10-11.5 with HC1. The solution is then incubated, e.g., at room temperature, for another 30 min to 24hr. Refolding efficiency can be improved with the addition of refolding agents such as sucrose, urea, 1,1-dimethylurea, DMSO or detergents such as CHAPS or sarkosyl. Preferably, 5% sucrose improves the refolding yield and eventual PTH(l-34) yield. Once refolding has reached equilibrium, Tris can then be added to a final concentration of 25mM, and the pH adjusted to 7.0 to 9.0 with HC1 or NaOH. pH 8.0 is the preferred pH. A semi-purified source of recombinant or yeast-derived Ubp2 (Ubp2 protein: FP ratio ~1:500 ) is then added and the solution incubated for between 1 and 48 hours at 20 to 35C. Preferred conditions are 16 hours at 25C. Following cleavage of the FP, the pH is adjusted to ~6.0 with HC1. A portion of the misfolded, aggregated and uncleaved FP protein precipitates. The solution is clarified using standard techniques such as filtration or centrifugation. The PTH(l-34) is extracted and purified from the clarified solution by standard chromatographic techniques used in the purification of peptides, e.g., cation-exchange and reverse-phase chromatography.

The ion exchange chromotography columns used in the invention may be comprised of any of those ion-exchange resins readily available. For example, exchange resins based on cellulose, dextran or agarose, may be used. The resins may be adapted to anionic or cationic exchange resins by the addition of appropriately charged functional groups according standard methodology. Anion exchange columns (eg, DEAE (diethylaminoethyl) or Q-type (quaternary arnine)) or cation exchange columns (eg, CM (carboxymethylcellulose) or S-type (sulphonated)) are most frequently used in the art of protein/peptide purification.

Appropriate columns for use in the invention may be prepared at the bench or alternatively purchased in ready to use form from commercial sources. Where one is required to prepare of a column standard proceedures used in the art may be followed; see, for example Choudhary GA & Horvath C (1996), Methods Enzymol 270, 47.

Following suitable preparation of the ion exchange column the products of the cleavage reaction in suitable form may be processed through the column according to standard procedures. Generally this involves a first step of loading the products onto the column to allow adsorption of one or more of L-Ubi, L-Ubi-fusion protein, and the protein or peptide of interest to the resin of the column. Subsequently, a wash buffer containing a low concentration of NaCl may be fed to the column to remove any nonspecific matter from the column. The concentration of NaCl in the wash buffer is selected to remove weakly bound protein, but not the protein or peptide of interest. Next, an appropriate elution buffer is fed through the column to effect differential movement of each of L-Ubi, L-Ubi- fusion protein, and the protein or peptide of interest through the column with the Uquid phase. Eluent is collected and the protein or peptide of interest collected or retrieved therefrom according to standard proceedures used in the art of protein/peptide purification. It will be appreciated that the column may be gravity fed or solutions may be fed thereto under positive pressure.

The nature of the buffers used in ion exchange chromatography according to the invention may vary depending on the characteristics of the protein/peptide of interest (pi and net charge, for example), the general nature and net charge of the leader sequence employed, and the type of resin/functional groups utilised, for example. Buffers commonly used in ion exchange chromatography are considered appropriate for use in the present invention; for example Tris (pH 7-9), Phosphate (ph 6-8), and Acetate (pH 4-6) buffers.

Due to the nature of ion exchange chromatography it will be understood that it is the salt concentration and pH conditions of the buffers which are primarily used in each step to control adsorption, wash stringency, and elution. Such conditions may be chosen based on knowledge of the net charge of the protein/peptide of interest, the L-Ubi and uncleaved L- Ubi-fusion proteinpeptide and the pis thereof. Information of the net charge of the protein/peptide of interest may be found in the literature in the form of electrophoresis or electrofocusing data. Where such data is not available one may estimate the net charge thereof by using software such as DNAsis (Hitachi Corp). Where the pi of a particular protein/peptide is unknown one may employ techniques standard in the art to estimate it; for example by employing electrophoretic separation perpendicular to a stable pH gradient. Where no useful information is immediately available one may utilise the high throughput ion exchange FPLC systems available to conduct optimisation assays to rapidly deteπnine the most appropriate conditions.

In relation to the present invention, the inventors believe that the pH of the ion exchange operation does not significantly affect the relative separation of the protein/peptide of interest from uncleaved L-Ubi-fusion protein/peptideprotein/peptides/peptides. However the amount of displacing salt, (e.g; NaCl), required to displace bound protein/peptideprotein/peptides/peptides from the ion exchange support will vary slightly with pH.

It will be appreciated that while the present invention relates to the use of ion exchange chromatography in the purification of a protein or peptide of interest such chromatography may be combined with other purification steps. Such other purification steps may include reverse-phase chromatography, hydrophobic chromatography, gel filtration, affinity chromatograhpy, dialysis and ultrafiltration, for example. Similarly, two or more sequential ion exchange chromatography steps may be employed. Such additional purification steps may occur prior to or post purification via an ion exchange column.

In addition to improved purification characteristics the inventors have suprisingly found that the inclusion of a leader sequence according to the invention may also improve the yield of proteins or peptides of interest produced by bacterial expression. The economic production of some proteins or peptides of interest in commercially useful amounts by bacterial expression has not previously been possible. Surprisingly the addition of a leader sequence, which may be charged, to the N-terminus of a Ubi-fusion protein/peptide improves yields to levels that are commercially and therapeutically useful. For example, adequate yields of Ubi-E2 IR for the production of E21R for therapeutic applications have been difficult to obtain. However addition of a leader sequence results in significant increases in production levels of the Ubi-E21R fusion protein. Such improved production levels are exemplified herein below.

The inventors contemplate that such improved production of Ubi-fusion protein will be so whether production relies on expression as inclusion bodies, protein or peptide expressed in the periplasmic space or secreted into culture media.

When making PTH(l-34) the following leader-Ubi-PTH(l-34) variants may be suitable. The part of the sequence shown in bold is the leader. The part of the sequence shown in italics is ubiquitin. PTH(1 -34) is shown in underline.

The (dm) refers to the deletion of the first amino acid of Ubi.

L8D-ubi-PTH(l-34):

MKEVKSlΛ>hΕ,FiMQIFVKTLTGKTπiEVESSDTIDNVKSKIQDKEGIPPDQQRπFAG KQLEDGRTLSDYNIQKESTLHL I-_ /.GGSVSEIOLMHNLGKHLNSMERNEWLRKK LODVHΝF L8D-(dm)ubi-PTH(l-34):

MKEyKS OVEFlQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGK QLEDGRTLSDYNIQKESTLHLVLRLRGGSVS OLMHNLGKKU^S ERVEWLRKKL ODVHΝF

K5D-(dm)ubi-PTH(l -34) :

MKEVOSlΛΛ EmQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGK QLEDGRTLSDYNIQKESTLHL FEi- RGGSVSEIOLMHΝLGKHLΝSMERVEWLRKKL ODVHΝF D8N-(dm)ubi-PTH(l-34):

MKEVKS1.NLEFIQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGK OEE_-)G^rES _W/O_^SrEHI E_-ZRGGSVSEIOLMHNLG-αiLNSMERVEWLRKKL ODVHNF

EXAMPLES Example 1 (hGH) Ubi-hGH was produced by bacterial expression as inclusion bodies (IBs) according to standard techniques (Kane JF & Hartley DL (1988) TIBiotech 6, 95). L-Ubi-hGH IBs were also produced, where L contained one Asp (MFATSSSTGNDG) or two Asp residues (MFADNDG). IBs were isolated and redissolved, and Ubi-hGH or L-Ubi-hGH preparations were refolded in vitro using standard methods (Fischer B et al (1993) Biotech Bioeng 41, 3). Routinely the refolding process resulted in -50% of Ubi-hGH or L-Ubi- hGH in correctly folded form, and available for cleavage with UbP. Cleavage was conducted by incubation in vitro with UbP2 at 30°C for 16 h in a buffer containing 25 mM Tris-HCl pH 8.0., to produce a mixture of natural hGH, Ubi or L-Ubi and uncleaved Ubi- hGH or L-Ubi-hGH. Natural hGH was purified from the other components of the cleavage reaction using a series of chromatographic steps.

• A first anion exchange chromatography step to remove the majority of uncleaved Ubi-hGH or L-Ubi-hGH (the majority of uncleaved Ubi-hGH or L- Ubi-hGH is aggregated, and does not bind), and Ubi or L-Ubi. • Hydrophobic Interaction Chromatography to remove essentially all remaining free Ubi, and reduce uncleaved Ubi-hGH or L-Ubi-hGH to -0.5%.

• A final (polishing) anion exchange step.

Specifically, the cleavage reaction solution in a buffer of Tris-HCl pH 8.0 (Buffer A) was loaded onto a column of Q-Sepharose Fast Flow (FFQ-Pharmacia) at a rate of 40 g of total protein per litre of FFQ. The flow rate was 100 mL/min (60 cm/h). At the completion of loading loosely bound protein, including the majority of L-Ubi, were washed off the column with Buffer A containing 70 mM NaCl. The n-hGH was subsequently removed with Buffer A containing 150 mM NaCl.

Hydrophobic Interaction Chromatography was performed by loading the 150 mM NaCl FFQ fraction onto a phenyl-Sepharose High Performance (Pharmacia) column pre- equilibrated in Buffer A containing 150 mM NaCl and 500 mM (NH )₂SO₄. Prior to chromatography the n-hGH-contaming FFQ fraction was adjusted to 500 mM (NH )₂SO (final concentration) by the addition of 1/6 volume of 3.5 M (NI_₄)_SO₄. The solution was loaded onto the column at a rate of 10 g of total protein per Utre of resin. The flow rate was 100 mL/min (60 cm/h). At the completion of loading, the column was washed with 6 column volumes of Buffer A containing 150 mM NaCl and 500 mM (NH )₂SO₄ to remove all traces of L-Ubi. The n-hGH was removed from the column by elution with Buffer A.

The final olishing) anion exchange step was carried out by loading the phenyl-Sepharose n-hGH fraction (previously desalted on a Sephadex G-25 column equilibrated in Buffer A) onto a Source Q (Pharmacia) column equilibrated with Buffer A, at a rate of 15 g of total protein per litre of resin. The flow rate was 30 mL/min (90 cm/h). At the completion of loading, the column was eluted with a 15 column volume gradient of 0-150 mM NaCl in Buffer A.

The elution profiles from the (polishing) anion exchange chromatography step are shown in Figure 1. Results show that n-hGH and Ubi-hGH co-elute at 19% Buffer B (buffer A containing 95 mM NaCl).

Addition of a leader containing a single Asp or two Asp residues displaces elution of the uncleaved fusion protein to 26% B and 32% B respectively. Accordingly, addition of a leader containing a single Asp or two Asp residues results in clear separation of uncleaved fusion protein from natural hGH on anion exchange chromatography, allowing preparation of purified natural hGH, essentially free of uncleaved fusion protein Example 2 (E21R)

Ubi-E21R and L-Ubi-E21R were produced by bacterial expression as IBs, where L contained one Asp residue (MFATSSSTGNDG). IBs were isolated and redissolved, and fusion proteinwere refolded in vitro using standard methods (Fischer B et al (1993) Biotech Bioeng 41, 3) as above. Routinely the refolding process resulted in ~[30]% of L- Ubi-E21R in correctly folded form, and available for cleavage with UbP. Cleavage was conducted by incubation in vitro with UbP2 as detailed in Example 1.

The purification procedure involved the use of anion-exchange chromatography to extract the protein from the refolding/cleavage solution. The majority of the aggregated forms of misfolded fusion protein passed through the column during loading of the solution. Some free Ubi or L-Ubi also passed through the column. The column was washed with a buffer containing 70 mM NaCl to remove the remaining free Ubi or L-Ubi. Correctly processed E21R was released from the column by washing the column with a buffer containing 200 mM NaCl. This step also removed some uncleaved fusion protein The E21R protein was further purified and resolved from uncleaved fusion protein on a high resolution anion- exchange column (e.g. Source 30Q - Pharmacia)- Using a gradient of NaCl, E21R was released from the column at 30%B whereas uncleaved fusion protein was released at 33%B.

Comparison of IB size ofL-Ubi-E21R and Ubi-E21R.

The size of IBs produced is dependent on the efficiency of expression, and is a good indicator of the yield of expressed protein In the present example there was insufficient Ubi-E21R produced for IB measurement. An L-E21R measurement of <0.45 μm was observed and a measurement of -0.57 μm for L-Ubi-E21R.

Purification

The yield of leaderless Ubi-E21R was not sufficient for purification of E21R. The predicted elution profile of Ubi-E21R during anion exchange chromatography was determined from its theoretical pi value. From this example it is noted that addition of a charged leader sequence to the N-terminus ofUbi-E21R:

• aUows the expression of L-Ubi-E21R, and the subsequent production of E21R in commercially useful yields.

• allows separation of E21R from uncleaved L-Ubi-E21R by anion exchange chromatography.

Example 3 (Expression of L8D-(dm)ubi-PTH(l-34) in E. coli MM294 ΔompT

1. Construction of human PTH(l-34) coding sequence

The entire coding region of PTH(l-34) was generated by amplification of the two overlapping oligonucleotides, 'PTH Upper' and 'PTH Lower'.

PTH Upper;

CAA GAG CCG CGG TGG TTCCGT TTC CGA AAT CCA ACT GAT GCA TAA CCT GGGTAAACATCT GAA CTC CAT GGAACGT

PTH Lower;

AAC AGC AAGCTTAGA AGT TAT GCA CAT CTT GCA GTT TCT TAC GCA GCCATT CCACAC GTTCCATGGAGTTCAGATG

1. Cloning PTH(l-34) PCR product into L8D-(dm)-ubi fusion vector, pBRE435

Kspl and HindHI treated PTH PCR product was ligated to similarly treated pBRE435 expression vector. Following desalting, an aliquot of the ligation reaction was transformed into electrocompetent E. coli DH5α cells. A plasmid, pBRE450, containing the PlΗ(l-34) coding region fused to L8D-(dm)-ubi was identified and confirmed by restriction analysis and subsequent sequencing analysis.

2. Transformation of pBRE450 into E. coli expression host

Plasmid pBRE450 was electroporated by standard methods into an E. coli K12 strain such as MM294 ΔompT. Transformants were selected on Luria agar containing ampiciUin.

3. Production of L8D-(dm)-ubi-PTH(l-34)

Expression of the L8D-(dm)ubi-PTH(l-34) fusion protein for purposes of PTH(l-34) isolation was performed by seeding 10 L minimal defined media with an inoculum of MM294 ΔompT(pBRE450) in a 22-L bioreactqr. Following overnight grow, the culture was induced at an OD600 of -40 with 10 ml 250 mM IPTG (final concentration

250μM ) for five hours. The culture was then passed six times through a 15M Homogenizer set at 9000 psi.. The homogenate was centrifuged at 8500 rpm for 10 minutes and the supernant discarded and the pellet resuspended in 4.25 L water and homogenized once at 9000 psi. The final pellet, following centrifugation at 4500 rpm for 20 minutes, was stored at

-20°C.

4. Refolding and Cleavage of L8D-(dm)ubi-PTH(l-34) and Extraction of intact PTH (1-34) Inclusion bodies (IBs) were refolded using a low-pH-method. That is, the IBs were solubiUsed in 15mM HC1 for lhr. The pH was adjusted to 3.5, using NaOH, and samples were incubated at room temperature for another lhr. Sucrose and Tris were added and dissolved to the final concentrations of 5% and 25mM respectively. The pH was then adjusted to 8.0. Sample was incubated overnight with a Ubp2 preparation (Ubp2: protein ratio: -1 :500 ). The pH was then adjusted to 6.0 at which the recombinant protein was precipitated. Supernatant was collected and purified by cation-exchange and reverse-phase chromatography.

It can be seen from the description provided herein that the addition of a leader sequence to a Ubi-fusion protein according to the invention overcomes the difficulty of separating a required protein or peptide from its uncleaved Ubi-fusion protein, and is applicable to the purification of a large number of protein or peptides from their uncleaved Ubi-fusion protein, including, but not restricted to those in Table 1. In addition the addition of the leader sequence to a Ubi-fusion protein/peptide has advantages in increasing the yield of fusion protein/peptide expressed in recombinant cells, especially bacterial cells.

The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent without departing from the scope of the invention. Furthermore, titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as Umiting the scope of the present invention.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour.

Throughout this specification unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

BRESAGEN LIMITED 11. November 2003

Claims

1. A fusion protein comprising Ubiquitin (Ubi) fused to the N-terminus of a protein or peptide of interest, and a leader sequence (L) comprising one or more a ino acids, said leader sequence being fused to the N-terminus of the Ubi.

2. A fusion protein as claimed in claim 1 wherein the leader sequence (L) comprises between 5 and 100 amino acids.

3. A fusion protein as claimed in any one of claims 1 or 2 wherein the leader sequence is chosen from the group comprising:

MFATSSSTGNDG;

MFATSSSTGDDG; MFATSSSDGDDG;

MFATSDSDGDDG;

MFATSSDTDNDG;

MFATDSDTDNDG;

MFADNDG; MFATSSSTGND;

MPATSSSTGNTMKEVKSLLLEFI;

MAPTSSSTGNTMKEVKSLLLDLQLLLEKVKNPENRKLSRMHTFEFI;

MKEVKSLLLEFI;

MKEVDSLLLEFI; MKEVKSLDLEFI;

MKEVDSLDLEFI;

M_OZVKSLLLDLQLLLEKVKNPENRKI.SRMHTFEFI; and

MKEVKSLLLDLQLLLEKV-CNPENRKLSRMHTFDFYVPKVNATEFI.

. A fusion protein as claimed in any one of claims 1 to 3 wherein the protein or peptide of interest is any protein or peptide which has at least 5 amino acids, more preferably at least 10 amino acids.

5. A fusion protein as claimed in claim 4 wherein the protein or peptide of interest is chosen from the group comprising:

Glucagon;

Relaxin;

Parathyroid hormone (PTH), PTH(l-34) and other PTH fragments; Amyloid B-protein fragments ;

Leptin or Leptin fragments (eg 22-56);

B-endorphin fragment;

Galanin;

Somatostatin; Brain natriuretic peptide;

Endothelin 2;

Endothelin 1;

Inhibin-like peptide;

HIV envelope fragments; Conotoxins;

Antibodies or antibody fragments;

Factor XHIa fragments; human growth hormone (hGH);

E21R; and IGF-1.

6. A fusion protein as claimed in claim 5 wherein the protein or peptide of interest is hGH, E21R, PTH, PTH(l-34) or IGF-1.

7. A fusion protein as claimed in claim 5 wherein the protein or peptide of interest is PTH(l-34).

8. A fusion protein as claimed in claim 5 wherein the protein or peptide of interest is IGF-1.

9. A fusion protein as claimed in any one of claims 1 to 8 wherein the leader sequence

(L) contains one or more charged residues.

10. A fusion protein as claimed in claim 9 wherein the charged residues are either: Asp and/or Glu; or Arg and/or Lys.

11. A fusion protein as claimed in claim 7 where the leader sequence is MKEVKSLDLEFI, MKEVDSLLLEFI, or MKEVKSLNLEFI.

12. A method for the production of a protein or peptide of interest comprising at least the steps of: a) producing in a host cell a fusion protein comprising Ubiquitin fused to the N-terminus of a protein or peptide of interest, and a leader sequence (L) comprising one or more amino acids, said leader sequence being fused to the N-terminus of the Ubi; b) conducting a cleavage reaction in which said fusion protein is contacted with a ubiquitin protease (UbP) to at least partially cleave the ubiquitin from the protein or peptide of interest; purifying the protein or peptide of interest from the products of said cleavage reaction.

13. A method as claimed in claim 12 wherein in step a) of said method, said fusion protein is formed as insoluble inclusion bodies, and the further steps of i) isolating the inclusion bodies from the host cell, ii) redissolving the inclusion bodies, and iii) refolding the fusion protein are conducted prior to steps b) and c).

4. A method as claimed in claim 12 wherein the leader sequence contains one or more charged residues and step c) requires the sub-steps of a) contacting the products of said cleavage reaction with an ion exchange column, b) selectively eluting the protein or peptide of interest from the column, and c) capturing the protein or peptide of interest.

15. A method as claimed in claim 12 wherein the pi of the cleaved leader-ubiquitin molecule is sufficiently different to the pi of the protein or peptide of interest so as to aUow efficient separation.

16. A method as claimed in any one of claims 12 to 15 wherein the host ceU is prokaryotic, preferably E. coli K12 or E. coli B.

17. A method as claimed in any one of claims 12 to 16 wherein the leader sequence is chosen from the group comprising:

MFATSSSTGNDG;

MFATSSSTGDDG;

MFATSSSDGDDG;

MFATSDSDGDDG; MFATSSDTDNDG;

MFATDSDTDNDG;

MFADNDG;

MFATSSSTGND;

MPATSSSTGNTMKΕVKSLLLΕFI; MAPTSSSTGNTMI ΕVKSLLLDLQLLLΕKVKNPΕNRKLSRMHTFΕFI;

MKΕVKSLLLΕFI;

MKEVDSLLLEFI:

MKEVKSLDLEFI

MKEVDSLDLEFI MKEVKSLLLDLQLLLEKVKNPE JRKLSRMHTFEFI; and MKEVKSLLLDLQLLLEKVKNPENRKLSRMHTFDFYVPKVNATEFI.

18. A method as claimed in claim 12 wherein the leader sequence is chosen from the group comprising:

MPATSSSTGNTMKEVKSLLLEFI;

MAPTSSSTGNTMKEVKSLLLDLQLLLEKVKNPENRKLSRMHTFEFI; MKEVKSLLLEFI; MKEVDSLLLEFI; MKEVKSLDLEFI;

MKEVDSLDLEFI;

MKEVKSLLLDLQLLLEKVKNPENRKLSRMHTFEFI; and MI EVKSLLLDLQLLLEKVKNPENRKLSRMHTFDFYVPKVNATEFI.

19. A method as claimed in claim 12 wherein the leader sequence is chosen from the group comprising:

MFATSSSTGNDG;

MFATSSSTGDDG;

MFATSSSDGDDG; MFATSDSDGDDG;

MFATSSDTDNDG;

MFATDSDTDNDG; and

MFADNDG.

20. A method as claimed in any one of claims 12 to 19 wherein said protein or peptide of interest is chosen from the group comprising: Glucagon; Relaxin; Parathyroid hormone (PTH), PTH (1-34) and other PTH fragments;

Amyloid B-protein fragments;

Leptin or Leptin fragments (eg 22-56);

B-endorphin fragment; Galanin;

Somatostatin;

Brain natriuretic peptide;

Endothelin 2;

Endothelin 1 ; Inhibin-like peptide;

HIV envelope fragments;

Conotoxins;

Antibodies or antibody fragments;

Factor XHIa fragments; human growth hormone (hGH);

E21R; and

IGF-1.

21. A method as claimed in claim 20 wherein said protein or peptide of interest is chosen from the group comprising hGH, E2 IR. PTH, PTH(1 -34) or IGF- 1.

22. A method as claimed in claim 21 wherein said protein or peptide of interest is PTH(l-34).

23. A method as claimed in claim 21 wherein said protein or peptide of interest is IGF- 1.

24. A method of claim 21 where said leader is selected from, MKEVKSLDLEFI, MKEVDSLLLEFI, or MKEVKSLNLEFI.

5. A protein or peptide produced according to a method of any one of claims 12 to 24.