Isolation, Renaturation, and Formation of
`Disulf ide Bonds of Eukaryotic Proteins
`Expressed in Escherichia coli as
`Inclusion Bodies
`
`Bernhard Fisher," Ian Sumner, and Peter Goodenough
`A FRC Institute of Food Research, Department of Protein Engineering, Earley
`Gate, Whiteknights Road, Reading RG2 2EE England
`
`Received March 12, 1992Mccepted August 72, 1992
`
`Expression of recombinant proteins in Escherichia coli of-
`ten results in the formation of insoluble inclusion bodies. In
`case of expression of eukaryotic proteins containing cys-
`teine, which may form disulfide bonds in the native active
`protein, often nonnative inter- and intramolecular disulfide
`bonds exist in the inclusion bodies. Hence, several methods
`have been developed to isolate recombinant eukaryotic
`polypeptides from inclusion bodies, and to generate native
`disulfide bonds, to get active proteins. This article summa-
`rizes the different steps and methods of isolation and rena-
`turation of eukaryotic proteins containing disulfide bonds,
`which have been expressed in E. coli as inclusion bodies,
`and shows which methods originally developed for study-
`ing the folding mechanism of naturally occurring proteins
`have been successfully adapted for reactivation of recombi-
`nant eukaryotic proteins. 0 1993 John Wiley & Sons, Inc.
`Key words: recombinant protein Escherichia coli inclusion
`body renaturation disulfide bond
`
`INTRODUCTION
`During the last 20 years, manipulation of DNA in vitro
`has developed from the transfer of genetic information
`between procaryotic organisms" to a technology that
`facilitates efficient and controlled production of proteins
`in foreign hosts. A significant feature of these develop-
`ments is the ability to express eukaryotic genes in pro-
`karyotes such as Escherichia ~ o f i . ~ ~
`The supply of many
`eukaryotic polypeptides which have potential clinical or
`industrial use is often limited by their low natrual avail-
`ability. Gene cloning and expression in E. cofi can pro-
`vide a more abundant source of these proteins.
`The expression of recombinant proteins in bacteria,
`however, often results in the formation of inactive pro-
`tein that accumulates intracellularly. The formation of
`inactive proteins in bacterial systems appears to be in-
`dependent of the type of the protein.63 These inactive
`protein species often associate to form insoluble protein
`aggregates called inclusion bodies (for review on inclu-
`sion bodies see refs. 56, 63, 65, 81, and 92). In cases of
`expression of eukaryotic proteins containing cysteines
`
`* To whom all correspondence should be addressed.
`
`Biotechnology and Bioengineering, Vol. 41, Pp. 3-13 (1993)
`0 1993 John Wiley & Sons, Inc.
`
`which are able to form disulfide bonds in the native ac-
`tive protein, often nonnative inter- and intramolecular
`disulfide bonds exist in the inclusion body polypeptide
`material as well as reduced cysteine residues.
`To obtain polypeptides, the insoluble protein pellets
`must be separated from the other cellular components
`usually by homogenization, washing, and centrifugation.
`Inactive pellets are then solubilized in denaturants, such
`as guanidine hydrochloride or urea, which unfold the
`protein. In most examples, reducing reagents are added
`to reduce the polypeptide cysteines to break existing di-
`sulfide bonds to yield monomeric peptide chains.
`The unfolded reduced protein must then be refolded.
`This includes the removal of denaturant and excess re-
`ducing reagent. As a result of renaturation the polypep-
`tide chain can fold into its native structure and the
`native disulfide bonds form.
`The main concepts in deciphering the folding code, a
`second translation of the genetic message, have been de-
`veloped from in vitro refolding studies using purified
`proteins isolated from their natural sources. The concept
`states that the folding of a polypeptide chain is a spon-
`taneous process, depending only on the amino acid se-
`quence in a given environment, to reach a lower energy
`conformation.6 This process is thermodynamically con-
`trolled and driven by the hydrophobic effect .Io4 Analysis
`of renaturation of many proteins has proved that, during
`refolding, denatured polypeptides go through different
`intermediates: (1) unfolded polypeptide; (2) nucleation
`of folding; (3) formation of regular structures; (4) mol-
`ten globule stage; (5) folded domains; (6) folded mono-
`mers; (7) subunit association (for a reviews on protein
`folding mechanisms see refs. 1, 8, 18, 19, 29, 33, 41,
`44-46, 50-52, 97, and 104). Stability of the native state
`relative to nonnative states depends also upon specific
`solvent conditions.
`A key reaction in refolding of reduced and denatured
`eukaryotic proteins is the generation of native disulfide
`bonds. During early studies of the renaturation of dena-
`tured polypeptides, analysis of the generated disulfide
`
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`bonds and their locations were central tools for elucidat-
`Several further chemicals have been introduced to
`ing the folding pathways of several proteins such as lyso-
`oxidize reduced proteins; e.g., dehydroascorbic acid for
`zyme, 1.2.5.72 ribonuclease,’ and bovine pancreatic trypsin
`the renaturation of serine proteases” and proinsulin.88
`i n h i b i t ~ r . ~ ~ ~ ~ ~
`None of these methods have been established as well-
`controlled, universally applicable protocols.
`During this time, several successful methods have
`been developed to study the generation of disulfide
`However, all these methods have been developed for
`bonds from reduced, denatured polypeptides.
`studying the folding mechanism of proteins. The experi-
`1. Air oxidation: In the air oxidation of reduced
`ments have been started using purified material obtained
`proteins, oxygen is apparently used as the electron ac-
`from natural sources, containing naturally occurring di-
`sulfide bonds. Proteins were denatured and reduced in
`ceptor for the oxidation of reduced cysteine residues to
`form disulfide bonds.’ Air oxidation is performed by
`purified systems followed by renaturation in analytical
`amounts.
`aeration of the refolding solution, or simple exposure of
`In contrast, biotechnology requires the renaturation
`the refolding buffer containing the reduced denatured
`of eukaryotic genes expressed in E. coli from inclusion
`protein to air. The main drawback of this method is the
`lack of precise control over the process. It has been
`bodies. The starting material differs mainly because the
`shown that air oxidation is accelerated in the presence
`protein source is an inactive, aggregated, and insoluble
`of
`to
`mol/L metal ions such as Cu2+.283,79 Air
`polypeptide material. It may possess native and non-
`oxidation may be more successful if trace amounts of
`native intra- as well as intermolecular disulfide bonds,
`thiolcompounds, such as 2-mercaptoethanol, are pres-
`as well unusual free cysteine residues. Although it can
`make up to 90% of all protein in the inclusion body, it is
`ent.” Using air oxidation, disulfide bond containing
`
`
`proteins such as ribon~clease~,~ and l y ~ o z y m e ~ ~ ~ ~ ~ ~ ~ have
`not a pure or homogenous source.
`been renatured.
`Several previous reviews analyzed the high-level ex-
`2. Formation of protein disulfide bonds starting from
`pression of recombinant proteins and the formation and
`properties of inclusion bodies in E. coli.39,56,63,65,81,92
`mixed disulfides: Using about lo-’ mol/L oxidized gluta-
`This
`thione, almost all the reduced cysteines of the denatured
`study summarizes the different steps and methods of iso-
`proteins are converted to mixed disulfides of protein-
`lation and renaturation of eukaryotic proteins containing
`cysteine and glutathione. After removal of excess de-
`disulfide bonds, which have been expressed in E. coli as
`naturant and free glutathione, low concentrations of
`inclusion bodies. Those proteins have been included in
`cysteine are added to displace the glutathione from the
`this review, which have been used as representative ex-
`protein-S-S-glutathione to initiate the formation of in-
`amples in previous reviews on protein expression and
`inclusion body formation,39,56,63,65,81,92
`tramolecular protein disulfide bonds. This method may
`but more recent re-
`be controlled by varying the ratio of added cysteine to
`sults have been added.
`the concentration of the mixed disulfides. Reoxidation
`of several serine proteases starting from mixed disulfides
`has been successfully d e ~ e l o p e d . ~ ~ , ~ ~ * ~ ~
`3. Glutathione reoxidation: An efficient system for
`renaturation of reduced proteins using a mixture of re-
`duced and oxidized glutathione has been introduced by
`Saxena and Wetla~fer.’~ At a ratio of about 10: 1 of re-
`duced to oxidized glutathione and a concentration of
`reduced glutathione of about loT3 mol/L, several dena-
`
`tured proteins such as l y s o ~ y m e , ~ ~ ~ , ~ ~ ribonuclease? and
`bovine pancreatic trypsin inhibitor 18s20 have been rena-
`tured, and the correct intramolecular disulfide bonds
`generated. This method is controlled by the concentra-
`tion of reduced and oxidized glutathione and their ratio
`to the protein-cysteine concentration.
`4. Reoxidation by dithiothreitol: Reoxidation of re-
`duced polypeptides by dithiothreitol has been most suc-
`cessfully developed by Creighton and co-workers 18*20 for
`studying the folding pathway of bovine pancreatic tryp-
`sin inhibitor. In a reaction of reduced protein-cysteine
`and oxidized dithiothreitol, an unstable intermediate of
`both is formed which dissolves with the release of re-
`duced dithiothreitol and the formation of intramolecular
`disulfide bonds. This reaction is strongly controlled by
`the concentration of added oxidized dithiothreitol.
`
`ISOLATION OF THE INCLUSION BODIES
`After fermentation of bacterial host cells, expressed gene
`products have to be isolated. Inclusion bodies seems to be
`very compact and table?^^^^*^^,^^.^^ They are localized in
`
`the ~ y t o p l a s m ~ ~ , ~ ~ ~ ~ ~ of the bacterial cells. To obtain inclu-
`sion bodies, E. coli cells were disintegrated by mechanic
`forces such as French
`homogeniza-
`tion,15,21.59,89 son~cat~on,23,30.34.43,46,53,57,58,76,78,6Z,67,9O,95,96,lOO
`or by a combination of lysozyme treatment and sonica-
`ion ~4,22,31,40,42,64,66,94,99,101,102
`There is no apparent corre-
`lation between the expressed protein and the isolation
`method. After disruption of E. coli cells, inclusion bod-
`ies are sedimented from the homogenate by low-speed
`centrifugation at 5,000 to 20,OOOg for about 15 min.
`In addition to the plasmid encoded protein, inclu-
`sion bodies can contain other proteins, such as the four
`subunits of RNA polymerase; some combination of
`the outer membrane proteins OmpC, OmpF, and
`OmpA; 16s and 23s rRNA; plasmid DNA; or other en-
`zymes .64,81,82 To purify inclusion bodies from adher-
`ing impurities they can be washed several times with
`buffer containing sucrose, Triton-100, deoxycholate or
`urea ~9,30,54,59,64,67.70,73,78,82,84,86,89,102
`The majority of pro-
`
`4
`
`BIOTECHNOLOGY AND BIOENGINEERING, VOL. 41, NO. 1, JANUARY 5, 1993
`
`Page 2
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`

`
`teins associated with the inclusion bodies, such as mem-
`brane proteins64 or kanomycin phosphotransferase,82
`have been substantially solubilized without solubilization
`of the polypeptide material of interest. Table I shows
`several examples of different washing media used for the
`purification of isolated inclusion bodies. Investigations
`showed that inclusion bodies containing different pro-
`teins resisted up to 5 mol/L urea.23*54,69*78
`
`SOLUBlLlZATlON OF INCLUSION BODIES
`After isolation of inclusion bodies the polypeptides have
`to be solubilized. In most cases, the addition of a re-
`ducing reagent is necessary to break existing nonnative
`intramolecular and intermolecular disulfide bonds.
`Tables I1 and I11 summarize several of the conditions
`used for the solubilization of recombinant proteins from
`inclusion bodies by use of either guanidine hydrochlo-
`ride or urea. However, less frequently, detergents such as
`sodium dodecylsulfate have been used to solubilize in-
`
`clusion b o d i e ~ . ~ ~ , ~ ’ 2-Mercaptoethanol as well as dithio-
`threitol are commonly used to reduce the proteins. In
`most reports on inclusion body solubilization and re-
`naturation of proteins, Tris buffer, at a concentration of
`about 0.1 mol/L, has been used. A comparison of the
`data presented in Tables I1 and I11 shows that solubiliza-
`tion and reduction is performed around pH 8. Concen-
`trations of about 8 mol/L urea or 6 to 8 mol/L guanidine
`HCI are widely used to solubilize proteins from inclusion
`bodies. Investigations on inclusion bodies containing
`proteins, such as bovine and human growth hormone
`and interleukin, showed that they resisted up to 5 mol/L
`urea -52,6932 In comparison to 2-mercaptoethanol, di-
`thiothreitol is used at lower concentrations, due to its
`stronger reducing potential.
`Results from Tables I1 and I11 show that there is
`no apparent correlation between the number of disul-
`
`Table I. Purification of inclusion bodies.
`
`Expressed protein
`
`Addition to washing buffer
`
`Refs.
`
`Eel GH/salmon GH
`89,84
`1 mol/L sucrose
`4% Triton X-100
`Eel GH/salmon GH
`89,84
`59
`2% deoxycholate
`Bovine GH
`5 mol/L urea
`Bovine GH
`82
`70
`0.1% Triton X-100
`Pro-urokinase
`73
`2.5% Triton X-100
`T-PA
`5 mol/L urea/2% Triton X-100 78,30
`T-PA
`0.375 mol/L sucrose
`95
`Human IL-2
`4 mol/L urea
`54
`Human IL-2
`Bovine pancreatic RNase 5% Triton X-100
`67
`Porcine phospholipase A2 1% Triton X-100
`9
`Human M-CSF
`2% Triton X-100
`102
`Horseradish peroxidase C 2 mol/L urea
`86
`0.5% Triton X-100
`64
`Prochymosin
`
`GH, growth hormone; T-PA, tissue-type plasminogen activator;
`IL, interleukin; RNase, ribonuclease; M-CSF, macrophage-colony
`stimulating factor.
`
`fide bonds in the native proteins and the method of
`solubilization/reduction. However, the temperature and
`time of exposure to denaturants vary drastically between
`different, and even within, the same protein. This is ob-
`viously caused by the incomplete knowledge and experi-
`ence in this field; most denaturation conditions have
`been selected empirically. Several proteins, such as im-
`munoglobulins, growth hormones, and tissue-type plas-
`minogen activator, have been solubilized by both urea
`and guanidine HCl. For most of these proteins, ad-
`vantages and disadvantages of different solubilization/
`reduction methods have not been analyzed in compara-
`tive studies to investigate their influences on the final
`yields. In addition, further research has to show if inclu-
`sion bodies are solubilized even at lower denaturant con-
`centrations. If different solubilization/reduction methods
`yield similar results for the same protein, this will enable
`selection of the method of choice to reduce expense in
`reagents, materials, and time.
`However, it was found for human interleukin-4 that
`solubilization in guanidine-hydrochloride increased the
`recovery of this protein, whereas, after solubilization in
`urea, no active peptide could be re~overed.~~
`Table IV shows additional methods that have been
`used successfully to isolate polypeptides from inclusion
`bodies without the use of denaturants and reducing re-
`agents. A comparison of the proteins listed in Table IV
`with proteins summarized in Tables I1 and I11 reveals
`that both recombinant bovine growth hormones and
`huma interleukins were isolated also from inclusion
`bodies by urea or guanidine HCI treatment. However, in
`several preparations, no reducing reagents were added to
`solubilize these proteins. Solubilization of several further
`proteins was achieved without the addition of reducing
`reagent (Tables I1 and 111). This points to the fact that
`these polypeptides existed in inclusion bodies without
`intermolecular disulphide bonds. It was found for re-
`combinant bovine growth hormone that this protein was
`stored in inclusion bodies in its complete, reduced form.59
`Further, several of these proteins, such as growth hor-
`mones and interleukins, have been renatured without a
`specific renaturationheoxidation method (see below).
`However, a correlation between the nature of these pro-
`teins and their specific renaturation behavior has not
`been analyzed so far.
`By contrast, when tissue-type plasminogen activator
`was isolated for the first time from inclusion bodies
`without a reducing reagent:’
`the yield was very poor, al-
`though the gene was expressed at high levels in E. coli.
`Later investigations on this enzyme showed that solu-
`bilization was achieved with high yields by adding
`2-mercaptoethanol or dithiothreitol, which demonstrated
`that this protein with a molecular weight of about 57,000
`and 34 cysteine residues forms high molecular weight ag-
`gregates held together by intermolecular disulfide bonds
`in the inclusion b ~ d i e s . ~ ’ , ” , ~ ~ , ~ ~ .
`
`FISCHER, SUMNER, AND GOODENOUGH: RENATURATION OF RECOMBINANT PROTEINS
`
`5
`
`Page 3
`
`

`
`Table XI. Solubilization of polypeptides from inclusion bodies by guanidine hydrochloride.
`
`Protein
`
`Immunoglobulin fragment
`Immunoglobulin chain
`Immunoglobulin fragment
`Creatine kinase
`T-PA
`T-PA
`T-PA
`T-PA mutant
`T-PA kringle-2 domain
`T-PA kringle-2 domain
`Low-molecular-weight urokinase
`Pro-urokinase
`Human serum albumin
`Insulin
`Insulin
`Chicken GH
`Bovine GH
`Bovine GH
`Eel GH
`Human IL-2
`Human IL-2
`Human IL-2
`Human IL-4
`Human IL-4
`
`Human IL-6
`Porcine phospholipase A2
`M-CSF
`Human angiogenine
`TGF-a
`IGF-I
`
`Number of
`disulfide bonds
`in active protein
`
`Guanidine HC1
`concentration
`(mol/L)
`
`Reducing reagent
`(mmol/L)
`
`5
`15
`2
`2
`17
`17
`17
`12
`3
`3
`6
`12
`17
`3
`3
`2
`2
`2
`2
`1
`1
`1
`3
`3
`
`2
`7
`9
`3
`3
`3
`
`6
`7.6
`7.6
`6
`7
`6
`6
`7
`7
`6
`5
`6
`7
`6
`7
`6
`6
`6
`5
`8
`7
`6
`6
`5
`
`6
`6
`7
`7
`7
`6
`
`DTT: 300
`2-ME: 100
`2-ME: 100
`D T T 100
`2-ME: 50
`DTE: 400
`DTE: 200
`2-ME: 100
`DTT: 200
`DTT: 5
`-
`2-ME: 50
`2-ME: 100
`2-ME: 140
`DTP: 1
`-
`-
`-
`-
`D T T 10
`-
`2-ME: 14
`2-ME: 14
`GSH: 2
`GSSG: 0.2
`-
`Na2S03: 300
`2-ME: 25
`2-ME: 100
`-
`2-ME: 10
`
`Period of
`solubilization Temperature
`(h)
`(“C)
`
`Refs.
`
`1
`1
`1
`1
`24
`3
`2.5
`24
`12
`12
`12
`12
`nr
`1
`nr
`nr
`nr
`80
`2
`1
`1
`2
`2
`
`1
`nr
`0.3
`4
`nr
`nr
`nr
`
`RT
`37
`37
`RT
`4
`25
`25
`4
`RT
`RT
`4
`RT
`4
`nr
`nr
`nr
`nr
`RT
`4
`37
`4
`RT
`RT
`
`RT
`nr
`4
`RT
`nr
`nr
`nr
`
`12
`13
`27
`8
`78
`73
`74
`30
`99
`42
`100
`70
`60
`35
`98
`34
`34
`59
`89
`96
`49
`95
`57
`
`53
`103
`9
`102
`23
`101
`76
`
`pH
`
`8.5
`8
`8
`8
`7.5
`8.6
`8.6
`7.5
`8
`8
`8
`8.5
`7.5
`nr
`7.9
`8.5
`8.5
`8
`8
`8.5
`7
`8
`8
`
`8
`8.3
`8
`7.5
`7.5
`nr
`nr
`
`GH, growth hormone; T-PA, tissue-type plasminogen activator; IL, interleukin; M-CSF, macrophage-colony stimulating factor; TGF-a,
`transforming growth factor-a; IGF, insulin-like growth factor; DTT, dithiothreitol; DTE, dithioerythritol; 2-ME, 2-mercaptoethanol;
`DTP, dithiopropanol; GSH, reduced glutathione; GSSG, oxidized glutathione; RT, room temperature; nr, not reported.
`
`Table 1x1. Solubilization of polypeptides from inclustion bodies by urea.
`
`Protein
`
`M-CSF
`Prochymosin
`Prochymosin
`Goat a-lactalbumin
`T-PA
`Peroxidase C
`EGF
`Immunoglobulin chain
`Immunoglobulin fragment
`Chicken lysozyme
`Bovine pancreatic RNase
`Salmon GH
`
`Number of
`disulfide bonds
`in active protein
`
`Urea
`concentration
`(mol/L)
`
`Reducing reagent
`(mmol/L)
`
`9
`3
`3
`3
`17
`4
`3
`15
`2
`4
`4
`2
`
`8
`8
`7.5
`8
`8
`8
`8
`7
`9
`8
`8
`7
`
`D T T 10
`-
`-
`DTT: 1
`-
`D T T 30
`D T T 1
`D T T 2
`2-ME: 20
`2-ME: 500
`2-ME: 25
`-
`
`Period of
`solubilization
`(h)
`
`Temperature
`(“C)
`
`0.5
`1
`nr
`nr
`nr
`1
`nr
`nr
`nr
`2
`1
`nr
`
`RT
`RT
`nr
`nr
`nr
`30
`nr
`nr
`nr
`40
`nr
`nr
`
`PH
`
`nr
`8
`7.5
`7.5
`8.5
`nr
`8.3
`8
`10.8
`8.6
`8
`8
`
`Refs.
`
`38
`64
`87,90
`58
`40
`86
`4
`11
`10
`43
`67
`84
`
`M-CSF, macrophage-colony stimulating factor; T-PA, tissue-type plasminogen activator; EGF, epidermal growth factor; GH, growth
`horman; RNase, ribonuclease; RT, room temperature; DTT, dithiothreitol; 2-ME, 2-mercaptoethanol; nr, not reported.
`
`RENATURATION AND REOXIDATION OF
`SOLUBILIZED POLYPEPTIDES
`After solubilization of inclusion bodies, the polypeptides
`are completely denatured and usually reduced. To obtain
`
`the native conformation and to generate correct disulfide
`bond formation, excess denaturant and reducing reagents
`have to be removed, and the polypeptide changed into a
`buffer under oxidizing conditions. The most frequently
`
`6
`
`BIOTECHNOLOGY AND BIOENGINEERING, VOL. 41, NO. 1, JANUARY 5, 1993
`
`Page 4
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`

`
`Table IV. Alternative methods of polypeptide isolation from inclusion bodies.
`
`Protein
`
`Human IL-1/3
`Human IL-2
`Human renin
`Bovine GH
`
`Number of disulfide
`bonds in active protein
`
`1
`1
`2
`2
`
`GH, growth hormone; IL, interleukin.
`
`Method of solubilization
`
`Refs.
`
`Extraction at acidic pH
`Incubation with Q-Sepharose
`Repeated vortex mixing
`Solubilization at pH 12
`
`55
`42
`83
`34
`
`protein concentrations the final yield of renatured active
`used way to reduce the concentration of denaturant and
`protein will be increaSed.12,’3,27,30,43,60,73,74,78
`reducing reagents is to dilute the polypeptide directly
`into the refolding bUffer.4,12,21,23,38,40,42,53,~,70,73,74,1~,10~ It
`Most successful methods for renaturationheoxidation
`of recombinant eukaryotic proteins, i.e., air oxidation,
`is also possible to dialyse the protein against different
`the glutathione renaturation system, and the reoxidation
`buffers’ 1,30,35,74,76,89,99 or to remove the chemicals by gel
`starting from mixed disulfides, have been used. In the
`filtration.86 Starting the refolding by dilution changes
`air oxidation system the reduced cysteine residues are
`the solvent properties surrounding the denatured pro-
`oxidized by oxygen and disulfide bonds are formed.
`tein instantly. This makes the process more kinetically
`Table V shows a summary of different conditions used
`dependent. By contrast, dialysis of the denatured protein
`for the reoxidation of recombinant proteins by this
`against decreasing denaturant concentrations allows the
`method. Process control during air oxidation should in-
`transformation to approach equilibrium conditions.
`clude dissolved oxygen control and precise control over
`Ideally, the renaturation procedures employed both
`oxidation catalyst concentration. However, no studies
`in research and industrial applications would use high
`have been reported in which an attempt was made to
`protein concentrations. However, a common observa-
`specifically control these variables.
`tion from renaturation experiments of nonrecombinant
`In the glutathione reoxidation system the formation
`proteins has been that the final yield of renatured pro-
`of the disulfide bonds is generated by the redox-couple
`tein decreases, sometimes dramatically, with increas-
`of reduced and oxidized glutathione. By varying the
`ing concentrations of solubilized protein undergoing
`ratio of reduced to oxidized glutathione in relation to
`renaturation; concomitantly, the percentage of insoluble
`the concentration of the protein-cysteine residues, the
`inactive protein aggregates increases.71993 This is due to
`optimal refolding conditions for each polypeptide can be
`the formation of unstable intermediate p r o d u ~ t s . ~ ’ , ~ ~ ~ ~ ~
`determined (Table VI). This makes the glutathione sys-
`Caused by hydrophobic interactions between the nor-
`tem more flexible in comparison with the air oxidation
`mally inaccessible core residues that become exposed
`system. To prevent air oxidation in the glutathione sys-
`on the surface of folding intermediates7’ aggregation
`tem, the buffer solution must be deaerated and kept
`occurs. As a result, refolding of nonrecombinant pro-
`under an inert gas such as nitrogen. Tables V and VI
`teins has been performed at a low protein concentra-
`show that in both renaturation systems the reoxidation
`tion of about
`mol/L corresponding to about 1 to
`is performed mainly between pH values of 8 and 9. The
`20 pg/m~.25,36.39,47.61,62,71,79,85,88,93 Although the renatura-
`temperature during renaturation and the period of re-
`tion of recombinant proteins is dependent on protein
`concentration, this has not been documented in publica-
`oxidation varies considerably. The ratio of reduced and
`tions to the same degree as has been done with purified
`oxidized glutathione used varies from 10 : 1 to 1 : 1 at con-
`centrations of reduced glutathione of about
`mol/L.
`natural proteins. It is likely though, that at these low
`
`Table V. Renaturation and reoxidation of recombinant proteins isolated from inclusion bodies by air oxidation.
`
`Protein
`
`Number of disulfide
`bonds to be formed
`
`Addit ion
`
`Human IFG-I
`Creatine kinase
`Human serum albumin
`Prochymosin”
`Human IL-6”
`Human IL-2
`Angiogenine
`Bovine GH”
`Pro-urokinase
`
`3
`2
`17
`3
`2
`1
`3
`2
`12
`
`1 mol/L urea
`6 mol/L GnHCl
`1.5 pmol/L Cu~S0.,/0.6 mol/L GnHCl
`
`6 mol/L GnHCl
`2.5 mol/L urea
`
`Temperature
`(“C)
`
`Time
`(h)
`
`Refs.
`
`37
`4
`4
`nr
`nr
`20
`4
`20
`15
`
`16
`12
`24
`0.5
`nr
`3
`24
`20
`24
`
`77
`8
`60
`64
`103
`96
`23
`59
`70
`
`PH
`
`9.0
`nr
`8.5
`10.7
`8.5
`8.5
`8.5
`8.0
`8.0
`
`IGF, insulin-like growth factor; IL, interleukin; GH, growth hormone; GnHC1, guanidine hydrochloride; nr, not reported.
`a Isolation from inclusion bodies without addition of a reducing reagent.
`
`FISCHER, SUMNER, AND GOODENOUGH: RENATURATION OF RECOMBINANT PROTEINS
`
`7
`
`Page 5
`
`

`
`Table VI. Renaturation and reoxidation of polypeptides isolated from inclusion bodies by the glutathione system.
`
`Protein
`
`Immunoglobulin chain
`Immunoglobulin fragment
`Immunoglobulin fragment
`Immunoglobulin fragment
`T-PA
`T-PA"
`T-PA
`T-PA
`T-PA mutant
`T-PA kringle-2 domain
`T-PA Kringle-2 domain
`M-CSF
`M-CSF
`Low-molecular-weight urokinase"
`Peroxidase C
`
`Human IL-4
`TGF-a"
`Chicken lysozyme
`Bovine RNase
`
`Number of
`disulfide
`bonds to
`be formed
`
`Glutathione
`
`Reduced
`(mmol/L)
`
`Oxidized
`(mmol/L)
`
`Addition
`
`15
`5
`5
`2
`17
`17
`17
`17
`12
`3
`3
`9
`9
`6
`4
`
`3
`3
`4
`4
`
`0.5
`4.0
`5.0
`0.5
`0.5
`1.0
`2.0
`-
`0.5
`10.0
`1.25
`2.0
`0.5
`2.0
`0.7
`
`2.0
`1.25
`1 .o
`5.0
`
`0.1
`-
`0.5
`0.1
`0.3
`0.1
`0.2
`10.0
`0.3
`1.0
`1.25
`1 .o
`0.2
`0.02
`-
`
`0.2
`0.25
`0.5
`5.0
`
`3 mmol/L DTE
`
`2.5 mol/L urea
`0.8 mol/L urea
`
`2 mmol/L DTT
`
`1 mol/L GnHCl
`1.2 mol/L GnHCl
`
`2 mol/L urea
`1 mol/L GnHCl
`2 mol/L urea/
`0.1 mmol/L DTT
`0.5 mol/L GnHCl
`1 mol/L GnHCl
`
`PH
`
`10.8
`8.2
`8.0
`10.8
`8.75
`nr
`10.5
`10.5
`8.75
`9.0
`8.0
`8.5
`8.5
`8.0
`8.0
`
`8.0
`9.0
`8.0
`8.0
`
`Temperature Time
`("C)
`(h)
`
`Refs.
`
`4
`10
`10
`4
`15
`30
`20
`20
`15
`RT
`RT
`4
`4
`4
`RT
`
`RT
`nr
`38
`25
`
`40
`150
`150
`40
`24
`2
`24
`24
`24
`48
`2
`96
`48
`36
`6
`
`4
`24
`1
`16
`
`11
`12
`12
`10
`78
`40
`73
`73
`30
`99
`42
`38
`102
`100
`86
`
`53
`101
`43
`67
`
`T-PA, tissue-type plasminogen activator; M-CSF, macrophase-colony stimulating factor; TGF-a transforming growth factor; IL, interleu-
`kin; RNase, ribonuclease; nr, not reported; GnHCI, guanidine hydrochloride; DTT, dithiothreitol.
`a Isolated from inclusion bodies without an addition of a reducing reagent.
`
`For most of the proteins that have been successfully re-
`e t h a n ~ l , ~ ~ , ~ ~ reduced glutathi~ne,'~'~~ or both reduced and
`
`
`natured it was found that an addition of 1 to 2 mol/L
`
`oxidized g l ~ t a t h i o n e . ~ * ' ~ ~ ~ ~ By disulfide shuffling, the
`urea or guanidine hydrochloride during renaturation in-
`more energetically stable native intramolecular protein
`creased the final yield of active protein and reduced the
`disulfide bonds are generated.
`reaggregat ion.
`Renaturation and reoxidation by disulfide interchange
`Recombinant proteins can also be renatured and na-
`refolding starting from mixed disulfides differs from air
`tive disulfide bonds formed from mixed disulphides:
`oxidation and the glutathione renaturation system: (1) It
`Mixed disulfides of polypeptide-cysteine residues and
`minimizes polypeptides from forming intermolecular
`glutathione, and polypeptide-cysteine-sulfinates, pre-
`disulfide bonded polymers. (2) It introduces a large num-
`pared by incubation of the peptides under denatur-
`ber of hydrophilic groups into the polypeptide which
`ing conditions with oxidized glutathione or Na-sulfite/
`raise the solubility of the denatured protein. (3) It en-
`Na-tetrathionate (Table VII), are renatured and re-
`ables the protein to be folded to its native conformation
`oxidized using different methods (Table VIII). After
`through a disulfide interchange rnechani~m.~~ However,
`formation of mixed disulfides, excess of reagent is
`formation of mixed disulfides by oxidized glutathione
`reduced by gel filtration or dialysis.9~'2~'3~22,31~35~73~74~103
`increases the expense of the process due to the cost of
`Disulfide interchange is initiated by an addition of low
`glutathione. Research should show if other disulfide sys-
`concentrations of cysteine?l dithi~threitol,'~ 2-mercapto-
`tems, such as cysteine and cystine, can replace glu-
`
`Table VII. Formation of mixed disulfides from polypeptides isolated from inclusion bodies.
`
`Protein
`
`Incubation
`
`Time
`(h)
`
`Temperature
`("C)
`
`PH
`
`Refs.
`
`Human IGF-I1
`0.1 mol/L Na-sulfite, 10 mmol/L Na-tetrathionate
`7 mol/L urea,
`7.4
`nr
`nr
`31
`8 mol/L urea,
`0.3 mol/L Na-sulfite, 65 mmol/L Na-tetrathionate
`Insulin
`9.0
`24
`RT
`35
`Phospholipase A2
`6 mol/L GnHC1, 0.3 mol/L Na-sulfite, 14 mmol/L NTSB
`7.5
`4
`9,22
`1
`Proinsulin
`6 mol/L GnHC1, 0.4 mol/L Na-sulfite, 12 mmol/L Na-tetrathionate
`9.0
`6
`RT
`98
`Immunoglobulin
`7 mol/L GnHCI, 0.2 mol/L Na-sulfite, 32 mmol/L Na-tetrathionate
`8.5
`16
`RT
`13
`Immunoglobulin
`6 mol/L GnHCl, 0.2 mol/L oxidized glutathione
`8.5
`3
`RT
`12
`T-PA
`6 mol/L GnHCI, 0.1 mol/L oxidized glutathione
`9.3
`3.5
`25
`74
`IGF, insulin-like growth factor; T-PA, tissue-type plasminogen activator; NTSB, disodium 2-nitro-5-thiosulfobenzoate; nr, not reported.
`
`8
`
`BIOTECHNOLOGY AND BIOENGINEERING, VOL. 41, NO. 1, JANUARY 5, 1993
`
`Page 6
`
`

`
`Table VIII. Renaturation and reoxidation of polypeptides containing mixed disulfides.
`
`Protein
`
`Human IGF-I1
`
`Human insulin
`Human insulin
`
`Human insulin
`
`Phospholipase Az
`
`Immunoglobulin
`
`Immunoglobulin
`
`T-PA
`
`Number of disulfide
`bonds to be formed
`
`Mixed disulfide
`form
`
`Method for reoxidation
`
`pH
`
`Temperature
`(“C)
`
`Time
`(h)
`
`Refs.
`
`3
`
`3
`3
`
`3
`
`7
`
`15
`
`5
`
`17
`
`Protein-S-sulfonate
`
`Protein-S-sulfonate
`Protein-S-sulfonate
`
`Protein-S-sulfonate
`
`Protein-S-sulfonate
`
`Protein-S-sulfonate
`
`Protein-S-glutathione
`
`Protein-S-glutat hione
`
`Disulfide interchanging
`by cysteine
`Air oxidation
`Disulfide interchanging
`by 0.4 mmol/L 2-ME
`Disulfide interchanging
`by DTT
`Disulfide interchanging
`by glutathione
`Disulfide interchanging
`by glutathione
`Disulfide interchanging
`by glutathione
`Disulfide interchanging
`by glutathione
`
`10.0
`
`9.0
`10.6
`
`10.5
`
`8.0
`
`10.8
`
`7.8
`
`8.5
`
`4
`
`RT
`0
`
`4
`
`RT
`
`4
`
`10
`
`RT
`
`16
`
`6
`4
`
`24
`
`24
`
`15
`
`150
`
`5
`
`31
`
`35
`98
`
`14
`
`9,22
`
`13
`
`12
`
`74
`
`IGF, insuline-like growth factor; T-PA, tissue-type ptasminogen activator; 2-ME, 2-mercaptoethanol; DTT, dithiothreitol.
`
`~~~~~~
`
`INCREASING THE YIELD OF RENATURED PROTEIN
`tathione. However, sulfonation by sodium sulfite is less
`expensive, and sulfonation can be performed directly
`Several methods have been used to increase the yield of
`from the inclusion b o d i e ~ . ~ , ~ ~ , ~ ~
`renatured protein by preventing aggregation during the
`There exist only a few investigations on renaturation
`refolding of nonrecombinant proteins. These include
`of the same protein but using different systems. The re-
`binding of proteins to CNBr-activated Sepharo~e,6~,~~
`re-
`sults showed that similar renaturation yields were ob-
`folding in the presence of high-molecular-weight poly-
`tained using the glutathione renaturation system and
`mers such as polyethylene-glycol l6 and performing the
`
`renaturation starting from mixed d i s ~ l f i d e s . ’ ~ ~ ~ ~ ~ ~ ~ How-
`renaturation in reversed m i ~ e l l e s . ~ ~ To increase the re-
`ever, the contribution of such important parameters as
`naturation yield of recombinant human insulin-like
`temperature and time of renaturation on the final yield
`growth factor-I, a fusion protein with the IgG-binding
`of active protein have to be investigated more intensively
`domain derived from staphylococcal protein A has been
`in further research.
`expressed