(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2004/0018586 A1
`(43) Pub. Date:
`Jan. 29, 2004
`ROSendahl et al.
`
`US 20040018586A1
`
`(54) METHOD FOR REFOLDING PROTEINS
`CONTAINING FREE CYSTEINE RESIDUES
`
`(76) Inventors: Mary S. Rosendahl, Broomfield, CO
`(US); George N Cox, Louisville, CO
`(US); Daniel H Doherty, Boulder, CO
`(US)
`Correspondence Address:
`SHERIDAN ROSS PC
`1560 BROADWAY
`SUTE 1200
`DENVER, CO 80202
`(21) Appl. No.:
`10/276,358
`(22) PCT Filed:
`May 16, 2001
`(86) PCT No.:
`PCT/US01/16088
`
`Related U.S. Application Data
`(60) Provisional application No. 60/204,617, filed on May
`16, 2000.
`Publication Classification
`(51) Int. Cl. ................................................. C12P 21/06
`(52) U.S. Cl. .......................................... 435/68.1; 435/69.4
`
`ABSTRACT
`(57)
`The present invention relates to novel methods for making
`and refolding insoluble or aggregated proteins having free
`cysteines in which a host cell expressing the protein is
`exposed to a cysteine blocking agent. The Soluble, refolded
`proteins produced by the novel methods can then be modi
`fied to increase their effectiveness. Such modifications
`include attaching a PEG moiety to form PEGylated proteins.
`
`Page 1
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`KASHIV EXHIBIT 1006
`IPR2019-00791
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`US 2004/0018586 A1
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`Jan. 29, 2004
`
`METHOD FOR REFOLDING PROTEINS
`CONTAINING FREE CYSTEINE RESIDUES
`
`FIELD OF THE INVENTION
`0001. The present invention relates generally to methods
`of making proteins and more Specifically to recombinant
`proteins containing at least one “free cysteine residue, i.e.,
`a cysteine residue that does not participate in a disulfide
`bond.
`
`BACKGROUND OF THE INVENTION
`0002 Protein therapeutics generally must be adminis
`tered to patients by injection. Most protein therapeutics are
`cleared rapidly from the body, necessitating frequent, often
`daily, injections. There is considerable interest in the devel
`opment of methods to prolong the circulating half-lives of
`protein therapeutics in the body So that the proteins do not
`have to be injected frequently. Covalent modification of
`proteins with polyethylene glycol (PEG) has proven to be a
`useful method to extend the circulating half-lives of proteins
`in the body (Abuchowski et al., 1984; Hershfield, 1987;
`Meyers et al., 1991). Covalent attachment of PEG to a
`protein increases the protein's effective size and reduces its
`rate of clearance from the body. PEGs are commercially
`available in Several sizes, allowing the circulating half-lives
`of PEG-modified proteins to be tailored for individual
`indications through use of different size PEGs. Other docu
`mented in vivo benefits of PEG modification are an increase
`in protein Solubility and Stability, and a decrease in protein
`immunogenicity (Katre et al., 1987; Katre, 1990).
`0003) One known method for PEGylating proteins
`covalently attaches PEG to cysteine residues using cysteine
`reactive PEGs. A number of highly Specific, cysteine-reac
`tive PEGs with different reactive groups (e.g., maleimide,
`vinylsulfone) and different size PEGs (2-40 kDa, single or
`branched chain) are commercially available. At neutral pH,
`these PEG reagents selectively attach to “free” cysteine
`residues, i.e., cysteine residues not involved in disulfide
`bonds. Cysteine residues in most proteins participate in
`disulfide bonds and are not available for PEGylation using
`cysteine-reactive PEGS. Through in Vitro mutagenesis using
`recombinant DNA techniques, additional cysteine residues
`can be introduced anywhere into the protein. The newly
`added “free” or “non-natural cysteines can serve as sites for
`the Specific attachment of a PEG molecule using cysteine
`reactive PEGs. The added “free” or “non-natural cysteine
`residue can be a Substitution for an existing amino acid in a
`protein, added preceding the amino-terminus of the mature
`protein or after the carboxy-terminus of the mature protein,
`or inserted between two normally adjacent amino acids in
`the protein. Alternatively, one of two cysteines involved in
`a native disulfide bond may be deleted or substituted with
`another amino acid, leaving a native cysteine (the cysteine
`residue in the protein that normally would form a disulfide
`bond with the deleted or substituted cysteine residue) free
`and available for chemical modification. Preferably the
`amino acid Substituted for the cysteine would be a neutral
`amino acid Such as Serine or alanine. For example, human
`growth hormone (hGH) has two disulfide bonds that can be
`reduced and alkylated with iodoacetamide without impair
`ing biological activity (Bewley et al., (1969). Each of the
`four cysteines would be reasonable targets for deletion or
`Substitution by another amino acid.
`
`0004 Several naturally occurring proteins are known to
`contain one or more “free” cysteine residues. Examples of
`Such naturally occurring proteins include human Interleukin
`(IL)-2 (Wang et al., 1984), beta interferon (Market al., 1984;
`1985), G-CSF (Lu et al., 1989) and basic fibroblast growth
`factor (bFGF, Thompson, 1992). IL2, Granulocyte Colony
`Stimulating Factor (G-CSF) and beta interferon (IFN-B)
`contain an odd number of cysteine residues, whereas basic
`fibroblast growth factor contains an even number of cysteine
`residues.
`0005 Expression of recombinant proteins containing free
`cysteine residues has been problematic due to reactivity of
`the free Sulfhydryl at physiological conditions. Several
`recombinant proteins containing free cysteines have been
`expressed cytoplasmically, i.e., as intracellular proteins, in
`bacteria Such as E. coli. Examples include natural proteins
`Such as IL-2, beta interferon, G-CSF, and engineered cys
`teine muteins of IL-2 (Goodson and Katre, 1990), IL-3
`(Shaw et al., 1992), Tumor Necrosis Factor Binding Protein
`(Tuma et al., 1995), Insulin-like Growth Factor-I (IGF-I,
`Cox and McDermott, 1994), Insulin-like Growth Factor
`binding protein-1 (IGFBP-1, Van Den Berg et al., 1997) and
`protease nexin and related proteins (Braxton, 1998). All of
`these proteins were predominantly insoluble when
`expressed intracellularly in E. coli. The insoluble proteins
`were largely inactive and needed to be refolded in order to
`regain Significant biological activity. In Some cases the
`reducing agent dithiothreitol (DTT) was used to aid solu
`bilzation and/or refolding of the insoluble proteins. Purified,
`refolded IL-2, G-CSF and beta interferon proteins are
`unstable and lose activity at physiological pH, apparently
`due to disulfide rearangements involving the free cysteine
`residue (Wang et al., 1984; Mark et al., 1984; 1985; Oh-eda
`et al., 1990; Arakawa et al., 1992). Replacement of the free
`cysteine residue in these proteins with Serine, resulted in a
`protein that was more stable at physiological pH (Wang et
`al., 1984; Mark et al., 1984; 1985; Arakawa et al., 1993).
`0006 A second known method for expressing recombi
`nant proteins in bacteria is to Secrete them into the periplas
`mic space or into the media. It is known that certain
`recombinant proteins Such as GH are expressed in a Soluble
`active form when they are Secreted into the E. coli peri
`plasm, whereas they are insoluble when expressed intracel
`lularly in E. coli. Secretion is achieved by fusing DNA
`Sequences encoding GH or other proteins of interest to DNA
`Sequences encoding bacterial Signal Sequences Such as those
`derived from the stII (Fujimoto et al., 1988) and ompA
`proteins (Ghrayeb et al., 1984). Secretion of recombinant
`proteins in bacteria is desirable because the natural N-ter
`minus of the recombinant protein can be maintained. Intra
`cellular expression of recombinant proteins requires that an
`N-terminal methionine be present at the amino-terminus of
`the recombinant protein. Methionine is not normally present
`at the amino-terminus of the mature forms of many human
`proteins. For example, the amino-terminal amino acid of the
`mature form of human GH is phenylalanine. An amino
`terminal methionine must be added to the amino-terminus of
`a recombinant protein, if a methionine is not present at this
`position, in order for the protein to be expressed efficiently
`in bacteria. Typically addition of the amino-terminal
`methionine is accomplished by adding an ATG methionine
`codon preceding the DNA sequence encoding the recombi
`nant protein. The added N-terminal methionine often is not
`removed from the recombinant protein, particularly if the
`
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`Jan. 29, 2004
`
`recombinant protein is insoluble. Such is the case with hCGH,
`where the N-terminal methionine is not removed when the
`protein is expressed intracellularly in E. coli. The added
`N-terminal methionine creates a "non-natural” protein that
`potentially can Stimulate an immune response in a human. In
`contract, there is no added methionine on hCGH that is
`Secreted into the periplasmic space using stI (Chang et al.,
`1987) or ompA (Cheah et al., 1994) signal sequences; the
`recombinant protein begins with the native amino-terminal
`amino acid phenylalanine. The native hCH protein Sequence
`is maintained because bacterial enzymes cleave the stI-hGH
`protein (or ompa-hGH protein) between the stII (or ompa)
`Signal Sequence and the Start of the mature hCH protein.
`0007 hCGH has four cysteines that form two disulfides.
`hGH can be secreted into the E. coli periplasm using stI or
`omp A Signal Sequences. The Secreted protein is Soluble and
`biologically active (Hsiung et al., 1986). The predominant
`Secreted form of hCGH is a monomer with an apparent
`molecular weight by Sodium dodecyl Sulfate polyacrylamide
`gel electrophoresis (SDS-PAGE) of 22 kDa. Recombinant
`hGH can be isolated from the periplasmic space by using an
`osmotic shock procedure (Koshland and Botstein, 1980),
`which preferentially releases periplasmic, but not intracel
`lular, proteins into the osmotic shock buffer. The released
`hGH protein is then purified by column chromatography
`(Hsiung et al., 1986). A large number of GH mutants have
`been Secreted into the E. coli periplasm. The Secreted mutant
`proteins were Soluble and could be purified using procedures
`similar to those used to purify wild type GH (Cunningham
`and Wells, 1989; Fuh et al., 1992). Unexpectedly, when
`Similar procedures were used to Secrete GH variants con
`taining a free cysteine residue (five cysteines, 2N-1), it was
`discovered that certain recombinant GH variants were
`insoluble or formed multimers or aggregates when isolated
`using Standard OSmotic Shock and purification procedures
`developed for GH. Very little of the monomeric GH variant
`proteins could be detected by non-reduced SDS-PAGE in the
`oSmotic Shock lysates. Insoluble or aggregated GH variants
`have reduced biological activities compared to Soluble,
`properly folded hCGH. Methods for refolding insoluble,
`Secreted Growth Hormone variants containing a free cyS
`teine residue into a biologically active form have not been
`described.
`0008 Alpha interferon (IFN-O2) also contains four cys
`teine residues that form two disulfide bonds. IFN-O2 can be
`Secreted into the E. coli periplasm using the St Signal
`sequence (Voss et al., 1994). A portion of the secreted
`protein is soluble and biologically active (Voss et al., 1994).
`Secreted, soluble recombinant IFN-O2 can be purified by
`column chromatography (Voss et al., 1994). When similar
`procedures were attempted to Secrete IFN-C2 variants con
`taining a free cysteine residue (five cysteines, 2N-1), it was
`discovered that certain of the recombinant IFN-C2 variants
`were predominantly insoluble or formed multimerS or aggre
`gates when isolated using Standard purification procedures
`developed for IFN-C 2. Insoluble or aggregated IFN-O2
`variants have reduced biological activities compared to
`soluble, properly folded IFN-O2. Methods for refolding
`insoluble, Secreted IFN-C2 variants containing a free cyS
`teine residue into a biologically active form have not been
`described.
`0009 Human Granulocyte Colony-Stimulating Factor
`(G-CSF) contains five cysteine residues that form two
`
`disulfide bonds. The cysteine residue at position 17 in the
`mature protein sequence is free. Perez-Perez et al. (1995)
`reported that G-CSF could be secreted into the E. coli
`periplasm using a variant form of the ompA Signal Sequence.
`However, very little of the ompa-CSF fusion protein was
`correctly processed to yield mature G-CSF. The percentage
`of correctly processed G-CSF could be improved by co
`expressing the E. coli dnaK and dinal proteins in the host
`cells expressing the ompa-G-CSF fusion protein (Perez
`Perez et al., 1995). Correctly processed, secreted G-CSF was
`largely insoluble in all E. coli strains examined (Perez-Perez
`et al., 1995). Insoluble G-CSF possesses reduced biological
`activity compared to soluble, properly folded G-CSF. When
`Similar procedures were attempted to Secrete wild type
`G-CSF, G-CSF variants in which the free cysteine residue
`was replaced with serine G-CSF (C17S), and G-CSF
`(C17S) variants containing a free cysteine residue (five
`cysteines, 2N-1) using the stII signal sequence, it was
`discovered that the recombinant G-CSF proteins also were
`predominantly insoluble. Methods for refolding insoluble,
`secreted G-CSF proteins into a biologically active form have
`not been described.
`0010 Human Granulocyte Macrophage Colony-Stimu
`lating Factor (GM-CSF) contains four cysteine residues that
`form two disulfide bonds. Libbey et al. (1987) and Green
`berg et al. (1988) reported that GM-CSF could be secreted
`into the E. coli periplasm using the ompA signal Sequence.
`Correctly processed, secreted GM-CSF was insoluble
`(Libbey et al., 1987; Greenberg et al., 1988). Insoluble
`GM-CSF possesses reduced biological activity compared to
`soluble, properly folded GM-CSF. When similar procedures
`were attempted to secrete GM-CSF variants containing a
`free cysteine residue (five cysteines, 2N--I) using the st
`Signal Sequence, it was discovered that the recombinant
`GM-CSF proteins also were predominantly insoluble. Meth
`ods for refolding insoluble, secreted GM-CSF proteins into
`a biologically active form have not been described.
`0011 U.S. Pat. No. 5,206,344 and Goodson and Katre
`(1990) describe expression and purification of a cysteine
`substitution mutein of IL-2. The IL-2 cysteine mutein was
`insoluble when expressed intracellularly in E. coli. The
`protein was Solubilized by treatment with a denaturing agent
`either 10% sodium dodecyl sulfate (SDS) or 8M urea and
`a reducing agent 100 mM dithiothreitol (DTT)), refolded
`and purified by size-exclusion chromatography and reversed
`phase HPLC. Expression and purification of cysteine
`muteins of IL-3 are described in U.S. Pat. No. 5,166,322.
`The IL-3 cysteine muteins also were insoluble when
`expressed intracellularly in E. coli. The proteins were Solu
`blilized with a denaturing agent (guanidine) and a reducing
`agent (DTT), refolded and purified by reversed phase HPLC.
`The purified IL3 cysteine muteins were kept in a partially
`reduced state by inclusion of DTT in the storage buffers.
`When the inventors used only a denaturing agent agent and
`a reducing agent (DTT) to denature and refold insoluble
`cysteine muteins of GH and G-CSF, it was discovered that
`the refolded proteins were heterogeneous, comprising mul
`tiple molecular weight Species. Similarly, when the inven
`tors denatured and refolded insoluble, Secreted IFN-O2
`cysteine muteins with only a denaturing agent and a reduc
`ing agent (DTT), undetectable levels of properly folded
`IFN-O2 cysteine muteins were obtained.
`
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`
`0012 Malik et al. (1992) and Knusli et al. (1992)
`described conjugation of wild tpe GM-CSF with amine
`reactive PEG reagents. The amine-PEGylated GM-CSF
`comprised a heterogeneous mixture of different molecular
`weight PEG-GM-CSF species modified at multiple amino
`acid residues (Malik et al. 1992; Knusli et al., 1992). The
`various amine-PEGylated GM-CSF species could not be
`purified from each other or from non-PEGylated GM-CSF
`by conventional chromatography methods, which prevented
`Specific activity measurements of the various isoforms from
`being determined. Clark et al. (1996) described conjugation
`of GH with amine-reactive PEGs. Amine-PEGylyated GH
`also was heterogeneous, comprising a mixture of multiple
`molecular weight Species modified at multiple amino acid
`residues. The amine-PEGylated GH proteins displayed sig
`nificantly reduced biological activity (Clark et al., 1996).
`Monkarsh et al. (1997) described amine-PEGylated alpha
`interferon, which also comprised multiple molecular weight
`Species modified at different amino acid residues. Amine
`PEGylated alpha interferon also displayed reduced biologi
`cal activity. Tanaka et al. (1991) described amine-PEGylated
`G-CSF, which also comprised a heterogeneous mixture of
`different molecular weight species modified at different
`amino acid residues. Amine-PEGylated G-CSF displayed
`reduced biological activity (Tanaka et al., 1991). Kinstier et
`al. (1996) described a PEGylated G-CSF protein that is
`preferentially modifed at the non-natural N-terminal
`methionine residue. This protein also displayed reduced
`biological activity (Kinstler et al. 1996).
`0013 Therefore, despite considerable effort, a need still
`exists for methods that allow an insoluble or aggregated
`protein containing one or more free cysteine residues to be
`refolded into a Soluble, biologically active form in high
`yield. The present invention Satisfies this need and provides
`related advantages as well. Similarly, a need also exists for
`methods of generating homogeneous preparations of long
`acting recombinant proteins by enhancement of protein
`molecular weight, such as by PEGylation.
`SUMMARY OF THE INVENTION
`0.014. The present invention generally relates to methods
`for obtaining refolded, Soluble forms of proteins having one
`or more free cysteine residues and which are expressed by
`a host cell in an insoluble or aggregated form. Such proteins
`include, but are not limited to, members of the Growth
`Hormone Supergene family, such as GH, IFN-O2, G-CSF
`and GM-CSF proteins, and anti-angiogenesis factors, Such
`as endostatin and angiostatin. The methods are generally
`accomplished by (a) causing a host cell to express a protein
`containing a free cysteine residue in an insoluble or aggre
`gated form; (b) lysing the cell; (c) Solubilizing the insoluble
`or aggregated protein in the presence of a denaturing agent,
`a reducing agent and a cysteine blocking agent; and (d)
`refolding the protein by lowering the concentrations of the
`denaturing agent and reducing agents to levels Sufficient to
`allow the protein to renature to a biologically active form
`Optionally, the soluble, refolded protein is isolated from
`other proteins in the refold mixture.
`0.015
`Suitable host cells include bacteria, yeast, insector
`mammalian cells. Preferably, the host cell is a bacterial cell,
`particularly E. coli.
`0016 Preferably, the soluble, refolded proteins produced
`by the methods of the present invention are recombinant
`
`proteins, especially cysteine variants or cysteine muteins of
`a protein. AS used herein, the terms "cysteine variant' and
`“cysteine mutein' are meant to encompass any of the
`following changes in a protein's amino acid Sequence:
`addition of a non-natural cysteine residue preceding the
`amino terminus of the mature protein or following the
`carboxy-terminus of the mature protein; Substitution of a
`non-natural cysteine residue for an existing amino acid in
`the protein, introduction of a non-natural cysteine residue
`between two normally adjacent amino acids in the protein;
`or Substitution of another amino acid for a naturally occur
`ring cysteine residue that normally form a disulfide bond in
`the protein. The methods are useful for producing proteins
`including, without limitation, GH, G-CSF, GM-CSF and
`interferon, especially alpha interferon, cysteine variants of
`these proteins, their derivatives or antagonists. Other pro
`teins for which the methods are useful include other mem
`bers of the GH Supergene family, the Transforming Growth
`Factor (TGF)-beta Superfamily, platelet derived growth fac
`tor-A, platelet derived growth actor-B, nerve growth factor,
`brain derived neurotophic factor, neurotrophin-3, neurotro
`phin4, Vascular endothelial growth factor, chemokines, hor
`mones, endostatin, angiostatin, cysteine muteins of these
`proteins, or a derivative or an antagonist thereof Cysteine
`muteins of heavy or light chains of an immunoglobulin or a
`derivative thereof are also contemplated.
`0017 AS used herein, the term “cysteine blocking agent”
`means any reagent or combination of reagents that result in
`the formation of a reversibly blocked free cysteine residue in
`a protein. Examples of useful cysteine blocking agents
`include, but are not limited to, dithiols Such as cystine,
`cyStamine, oxidized glutathione, dithioglycolic acid and the
`like, or thiols Such as cysteine, cysteamine, thioglycolic
`acid, and reduced glutathione. Preferably, thiols should be
`used in the presence of an oxidizing agent. Useful oxidizing
`agents include oxygen, iodine, ferricyanide, hydrogen per
`oxide, dihydroascorbic acid, tetrathionate, and O-io
`doSobenzoate. Optionally, a metalion Such as copper (Cu"
`or cobalt (Co") can be added to catalyze the oxidation
`reaction. Although not wishing to be bound by any particular
`theory, the inventors postulate that the cysteine blocking
`agent forms a mixed disulfide with the free cysteine residue
`in the protein, thus limiting possible disulfide rearrangments
`that could occur involving the free cysteine residue. The
`mixed disulfide Stabilizes the free cysteine residue, Signifi
`cantly enhancing the yield of properly folded, biologically
`active, Soluble protein. AS used herein, reducing agents Such
`as DTT and 2-mercaptoethanol are not considered cysteine
`blocking agents because they do not result in the formation
`of a reversibly blocked mixed disulfide with the free cys
`teine residue in the protein. DTT typically does not form
`mixed disulfides with cysteine residues in proteins due to a
`thermodynamically preferred intramolecular bond that
`forms upon oxidation.
`0018 Higher order dimeric and multimeric proteins
`formed by the covalent association of two or more of the
`refolded proteins via their free cysteine residues also within
`the present invention.
`0019. The present methods further include various meth
`ods of attaching a cysteine-reactive moiety to the refolded
`protein to form modified protein in which the cysteine
`reactive moiety is attached to the refolded protein through
`the free cysteine residue(s). An example of a useful cysteine
`
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`US 2004/0018586 A1
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`
`reactive moiety that can be attached to the refolded protein
`is a cysteine-reactive PEG, which can be used to form a
`PEGylated protein Such methods include (a) isolating the
`refolded protein having a free cysteine residue from other
`proteins in the refold mixture; (b) reducing, at least partially,
`the isolated, refolded protein with a disulfide-reducing agent
`and (c) exposing the protein to a cysteine-reactive moiety
`such as a cysteine-reactive PEG. Optionally, the modified
`protein can be isolated from unmodified protein. Examples
`of other useful cysteine-reactive moieties are cysteine-reac
`tive dextrans, cysteine-reactive carbohydrates, cysteine-re
`active poly (N-vinylpyrrolidone)S., cysteine-reactive pep
`tides,
`cysteine-reactve
`lipids, and cysteine-reactive
`polysaccharides.
`0020. The present invention further includes the soluble,
`refolded proteins and their derivatives, including PEGylated
`proteins, made by the methods disclosed herein. Such
`PEGylated proteins include monopegylated, cysteine Vari
`ants of GH, G-CSF, GM-CSF and alpha interferon proteins.
`Such PEGylated proteins also include cysteine variants of
`GH, G-CSF, GM-CSF and alpha interferon proteins modi
`fied with two or more PEG molecules, where at least one of
`the PEG molecules is attached to the protein through a free
`cysteine residue.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`0021. The present invention provides novel methods of
`preparing refolded, soluble forms of GH, IFN-O2, G-CSF
`and GM-CSF proteins that have at least one free cysteine
`residue and which are expressed by a host cell in an
`insoluble or aggregated form. The present invention can be
`used to prepare refolded, soluble forms of other members of
`the GH Supergene family that have at least one free cysteine
`residue and which are expressed by a host cell in an
`insoluble or aggregated form The present invention also can
`be used to prepare refolded, soluble forms of other types of
`proteins having at least one free cysteine residue and which
`are expressed by a host cell in an insoluble or aggregated
`form, including, but not limited to, anti-angiogenesis pro
`teins Such as endostatin and angiostatin. The invention
`further provides novel proteins, particularly recombinant
`proteins produced by these novel methods as well as deriva
`tives of Such recombinant proteins. The novel methods for
`preparing Such proteins are generally accomplished by:
`0022 (a) causing a host cell to express a protein
`having a free cysteine in an insoluble or aggregated
`form;
`0023 (b) lysing the host cell by chemical, enzymatic
`or physical means,
`0024 (c) solubilizing the insoluble or aggregated
`protein by exposing the protein to a denaturing
`agent, a reducing agent and a cysteine blocking
`agent, and
`0025 (d) refolding the protein by reducing the con
`centrations of the denaturing agent and reducing
`agent in the Solubilization mixture to levels Sufficient
`to allow the protein to renature into a Soluble,
`biologically active form.
`Optionally, the refolded, soluble protein can be
`0.026
`isolated from other proteins in the refold mixture. The
`
`methods and other embodiments of the present invention
`were described in detail in U.S. Provisional Application
`Serial No. 60/204,617, filed May 16, 2000. U.S. Provisional
`Application Serial No. 60/204,617 is incorporated herein by
`reference in its entirety.
`0027 AS identified above, the first step in these methods
`is to cause a host cell to express a protein having a free
`cysteine residue in an insoluble or aggregated form Suitable
`host cells can be prokaryotic or eukaryotic. Examples of
`appropriate host cells that can be used to express recombi
`nant proteins include bacteria, yeast, insect and mammalian
`cells. Bacteria cells are particularly useful, especially E. coli.
`Methods of causing a host cell to express a protein are well
`known in the art and examples are provided herein.
`0028. As used herein, the term “protein having a free
`cysteine residue” means any natural or recombinant protein
`or peptide that contains 2N-1 cysteine residues, where N
`can be 0 or any integer, and any natural or recombinant
`protein or peptide that contain 2N cysteines, where two or
`more of the cysteines do not normally participate in a
`disulfide bond. Thus, the methods of the present invention
`are useful in enhancing the expression, recovery and puri
`fication of any protein or peptide having a free cysteine,
`particularly cysteine added variant recombinant proteins
`(referred to herein as "cysteine muteins” or “cysteine vari
`ants”) having one or more free cysteines. Although the
`expression, recovery and purification of a natural protein
`having a free cysteine expressed by its natural host cell can
`be enhanced by the methods of the present invention, the
`description herein predominantly refers to recombinant pro
`teins for illustrative purposes only. In addition, the proteins
`can be derived from any animal Species including human,
`companion animals and farm animals. The proteins also can
`be derived from plant Species or microbes.
`0029. Accordingly, the present invention encompasses a
`wide variety of recombinant proteins, and cysteine variants
`of these proteins. These proteins include members of the GH
`Supergene family, and cysteine variants of these proteins.
`The following proteins (“collectively referred to as the GH
`Supergene family’) are encoded by genes of the GH Super
`gene family (Bazan (1990; 1991; 1992); Mott and Campbell
`(1995); Silvennoinen and Ihle (1996); Martin et al. (1990);
`Hannum et al. (1994); Blumberg et al., 2001): GH, prolactin,
`placental lactogen, erythropoietin (EPO), thrombopoietin
`(TPO), interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL7,
`IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL13, IL-15, IL19,
`IL-20, IL-TIF, MDA-7, AK-155, oncostatin M, ciliary neu
`rotrophic factor, leukemia inhibitory factor, alpha interferon,
`beta interferon, gamma interferon, omega interferon, tau
`interferon, granulocyte-colony Stimulating factor (G-CSF),
`granulocyte-macrophage colony Stimulating factor (GM
`CSF), macrophage colony stimulating factor (M-CSF), car
`diotrophin-1 (CT-1), Stem Cell Factor and the flt3/flt2
`ligand. It is anticipated that additional members of the GH
`Supergene family will be identified in the future through
`gene cloning and Sequencing. Members of the GHSupergene
`family have Similar Secondary and tertiary Structures,
`despite the fact that they generally have limited amino acid
`or DNA sequence identity. The shared structural features of
`members of the GH Supergene family, which are described
`in Bazan (1990; 1991; 1992), Mott and Campbell (1995) and
`Silvennoinen and Ihle (1996), allow new members of the
`gene family to be readily identified. Variants of these pro
`
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`US 2004/0018586 A1
`
`Jan. 29, 2004
`
`teins Such as the Selective IL-2 antagonist described by
`Shanafelt et al. (2000) also are encompassed by this inven
`tion.
`0030 The present methods also can enhance the expres
`Sion, recovery and purification of additional recombinant
`proteins, including members of the TGF-beta Superfamily.
`Members of the TGF-beta Superfamily include, but are not
`limited to, glial-derived neurotrophic factor (GDNF), trans
`forming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF
`beta3, inhibin A, inhibin B, bone morphogenetic protein-2
`(BMP-2), BMP-4, inhibin alpha, Mullerian inhibiting Sub
`stance (MIS), and OP-1 (osteogenic protein 1). The mono
`mer subunits of the TGF-beta Superfamily share certain
`structural features that allow other members of this family to
`be readily identified: they generally contain 8 highly con
`Served cysteine residues that form 4 intramolecular disul
`fides. Typically a ninth conserved cysteine is free in the
`monomeric form of the protein but participates in an inter
`molecular disulfide bond formed during the homodimeriza
`tion or heterodimerication of the monomer Subunits. Other
`members of the TGF-beta Superfamily are described by
`Massague (1990), Daopin et al. (1992), Kingsley (1994),
`Kutty et al. (1998), and Lawton et al. (1997), incorporated
`herein by reference.
`0031) Immunoglobulin (Ig) heavy and light chain mono
`merS also contain cysteine residues that participate in
`intramolecular disulfides as well as free cysteines (Roitt et
`al., 1989 and Paul, 1989). These free cysteines normally
`only participate in disulfide bonds as a consequence of
`multimerization events Such as heavy chain homodimerza
`tion,
`heavy chain-light
`chain
`heterodimerization,
`homodimerization of the (heavy chain-light chain) het
`erodimers, and other higher order assemblies Such as pen
`tamerization of the (heavy chain-light chain) heterodimers in
`the case of IgM. Thus, the methods of the present invention
`can be employed to enhance the expression, recovery and
`purification of heavy and/or light chains (or various domains
`thereof) of human immunoglobulins Such as for example
`IgG1, IgG2, IgG3, IgG4, IgM IgA1, IgA2, Secretory IgA,
`IgD and IgE, and cysteine variants of these proteins or
`fragments thereof. Immunoglobulins from other Species
`could also be Similarly expressed, recovered and purified.
`Proteins genetically fused to immunoglobulins or immuno
`globulin domains, as described in Chamow & Ashkenazi
`(1996), could also be similarly expressed, recovered and
`purified.
`0032. A group of proteins has been classed as a structural
`Superfamily based on the shared Structural motif termed the
`“cystine knot'. The cystine knot is defined by six conserved
`cysteine residues that form three intramolecular disulfide
`bonds that are topologically "knotted” (McDonald and Hen
`drickson, 1993). These proteins also form homo- or het
`erodimers and in Some but not all instances dime