CA2194177A1 - Process for folding of proteins like recombinant hirudin or epidermal growth factor - Google Patents
Process for folding of proteins like recombinant hirudin or epidermal growth factorInfo
- Publication number
- CA2194177A1 CA2194177A1 CA002194177A CA2194177A CA2194177A1 CA 2194177 A1 CA2194177 A1 CA 2194177A1 CA 002194177 A CA002194177 A CA 002194177A CA 2194177 A CA2194177 A CA 2194177A CA 2194177 A1 CA2194177 A1 CA 2194177A1
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- CA
- Canada
- Prior art keywords
- protein
- process according
- proteins
- correct
- folded protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF] (urogastrone)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
- C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to a process for the preparation of a biologically active and correctly folded protein in the presence of a denaturing agent like urea or guanidine hydrochloride and the separation of the correctly folded protein therefrom directly. This process can be applied, for example, in the renaturation of recombinant proteins like hirudin or epidermal growth factor.
Description
~ W0 96103425 r~ lmO
Process for foldlng of pro~efns like recomb~nant hfrudfn or eptdermal growth factor .
The invention relates to a process for the ~".; " 1 of a biulugi~Al'y active and correctly folded protein in the presence of a denaturing agent.
Correct folding of genetically unyi~ee~ud proteins i5 a major issue in protein science. The problem is particularly acute, e.g., in the case of disulfide-containing proteins, which often need to be denatured and refolded to become active. Disulfide fommation is an event of post-l,a, ' " ~àl ~ . It follows the pathway of protein folding and usually ûccurs faithfully in vivo. Hûwever, in the in vitro ~ I expuli,l":"l~, many prûteins are recovered in poor yields and some do not fold into native ~;UI If Ull )r at all. To overcome this problem, a number of compounds, such as the mixture of reduced I oxidized gluta-thione (Creighton, Methods Enzymol. 131 (1986), 83-106) and protein disulfide isomerase (PDI) (Morin, J. E. & Dixon, J. E. Methods Enzymol. 113 (1985), 541-547) have been routinely used to promote the fommation of disulfides. But despite their ~ r~ use, the precise Ille-,l,dnis", of their function remains undefined and their a,, " )s have been conducted in a manner of trfal and error.
A number of pl'" ~s have appeared which report refolding attempts for individualproteins produced in bacterial hosts, or which are otherwise in a denatured or non-native foml. Formation of a dimeric, biologically active human colony stimulating factor-1 ~CSF-1 ) after expression in E. coti is described in WO 88/8ûû3 and by Halenbeck et al.
Ciul~.,-l",ology, 7 (1989), 71û-715. The plu~~dul~s described involve the steps of initial ' ' " , of CSF-1 monomers isolated from inclusion bodies under reducing conditions in a chaotropic en~;.ulll~l~lll comprfsing urea or guanidine hyd~uulllulide, refolding which is achieved by stepwise dilution of the chaotropic agents, and final oxidation of the refolded molecule in the presence of a redox-system. In WO 8818849 a process for recovering ~ulllbilldlll interleukin-2 (IL-2) is disclosed, l;ha~ault~ ed in that IL-2 isolated from refractile bodies is denatured under reduc:ng condition.s with 6M guanidine hydlu~,lllulidu, the soluble IL-2 is oxidized by a controlled oxidation in the presence of Cu2+ ions, and the oxidized IL-2 refolded by reducing the uJllcelllldliull of the denaturant in the solution.
Interleukin-2 and interferon-13 have been refolded using SDS for s~-lll' , and Cu2' ions as oxidation promoters of the fully reduced protein (US-A-4572798). The process for WO g6103~2~; PCT/EI'9~/1}27~
". '~ 2 ~ 9 4 1 7 7 isolating l~")llli illdnl refractile proteins as described in US-A-4620948 involves strong denaturing agents to solubili~e the proteins, reducing conditions to facilitate correct folding and denaturant IU; Id,ellle ll in presence of air or other oxidizing agents to refomm the disulfide bonds. A method for renaturing unfolded proteins including cytochrome c, ovalbumin and trypsin inhibitor by reversibly binding of the denaturetf protein to a solid matrix and stepwise renaturing it by diluting the denaturant is disclosed in WO 8615809.
The foregoing references are merely It:jJI(~ I ' ".'C of a huge amount of literature deaiing with the refolding of non-native proteins derived from different sources. The man skiiied in the art on the other hand knows that the success of refolding e~z,i,,,u,,l~ cannot be predicted. Unsuccessful experiments are usually not reported. There is no certainty ~hat anyone of the reported refolding conditions would work at ail with a given denatured protein containing several cysteine residues and therefore, a number of i,,l,d,,,ulecular disulfide bonds, which are reguired for activity.
Guanidine hydluuilluliLid (GdmCI) and urea are the best known denaturants for unfolding and inactivating proteins. Although the r.~el,l,dnia", ol their actions remains to be fuliy understood, it is generally evident that they disrupt the non-covalent interactions which stabiii~e the native uullfvl~ t;~ . The detrimental effect of GdmCI and urea has also been illustrated during the folding of proteins, which usually leads to the fommation of inactive, scrambled species ~Haber and Anfinsen, J. i3iol. Chem. (1962), 237, 183g-1844, Weissman and Kim Science ~1991~, 253, 1386-1393). On the other hand~ denaturants are potent agents for solubilizing intractable proteins, such as immunoglobulins and membrane ~,u~ u~e~lO etc. For example, lUCUllliJilldlll proteins expressed in an G~/~er~ d cOa system often face this problem of prolein solubility. These proteins are frequently iound in insoluble inciusion bodies and require s~ 1 by strong denaturant as the essentiai step to refoid and generate the active w"I. . ~.
Surprisingiy it has now been found that in the presence of denaturant and under equilibrium conditions correct foided protein is fommed and can be isoiated therefrom directly. This finding greatly facilitate the plU;) .rdlioll of l~ulniJilla,~l protelns as these proteins can be solubilized using denatunng agents and isolated therefrom in their correct folded native ~;unloilllclLiull without removing the solubilizing denaturant. Additionally, the presence oi ~ WO 96/03425 PCTII~P95102720 3 - 2 1 9 ~ 1 7 7 high uunu~lllldliulls of denaturant usually inactivates proteases and makes an addition of protease inllibitors unrl~u~adiy.
Therefore, the current invention provides a process for the production of a correct folded protein or a salt thereofl ,.I,d,dulcri~ed in that the protein is treated with a buffer comprfsing a denaturant, and the correct folded protein is separated therefrom directly, wherein the denaturant is selected from the group consisting of guanidine l.~J~uchludde in a COllCt:ll' ' nfrom3to7Mandureainacu,)ce"t,~t;vl,from~ito10M.
The wording correct folded protein stands for a protein that is in the native culllulllldliull andJor shows a biological activity like the enzymatic activity or the binding property of the native protein.
The inventive process is applicable to any protein or protein fragment that has to be folded into a correct folded culllolll~dliull and that e~ldL,li~lles an equilibrium between a not correct and a correct folded CUIIIOIII~ ;JII in presence of the denaturant. This is usually the case for proteins that are not irreversibly denatured by the denaturant. The ability of a protein to establish said equilibrium can easily be monitored by standard methods that provide il~loi~lldliun on the folding of proteins in solution like NMR or circular dicroism.
The protein to be refolded by the inventive process rnay be from almost any source, a - special ulclludllllulll is not necessary but not excluded. For example, a lu~,ul~lbilldlll protein that is stored in the producing host in form of inclusion bodies can be refolded by simply separating the inclusion bodies from the rest of the cell debris, 5~111' ' ,9 the proteins of the inclusion bodies with denaturant and isolating the correct folded protein therefrom. In case the protein is not stored in foml of inclusion bodies it is possible to enrich the protein only to some extend using, e.g., a uluCi,uildliull step, solubilize the protein with the denaturant and isolate the pure and correct folded protein therefrom. For ~ ~u~,u~bina~l proteins or natural proteins that are not in a correct folded cu"iu", ~ after isolation it is possible to solubilize these protein after isolation in the denaturant and isolate the correct folded fraction therefrom.
WO 9~i/0342:~ rCT~ ;fO2720 4 - ~ 1 9 4 1 7 7 Examples for suitable proteins are hinudin, epiderma! growth factor, potato ~;dl~A~p~Jiiddse inhibitor (PCI) and bovine pancreatic trypsin inhibitor ~BPTI), IGF-1, C5a-antagonist, TGF-,~. Especially preferred proteins are hirudin and epidermal growth factor ~EGF).
The temm "hinudinU as used in this invention is intended to embrace all desulfatohinudin compounds described in literature or obtainable from a tldll~lu~ ed Illi~"uo~ya"l:,"~ strain containing DNA which codes for a desulfatohirudin or a derivative thereot. Such hinudins are, for example, desulfatohinudin derivatives HV1, HV2 and HV3 (PA), as well as other hinudinproteinsasdescribede.g.byM.Schar'etaLIFEBSLett.,255(1989),1û5-110)and EP-A-347376. It is to be understood that hirudin derivatives or shorter fragments having hinudin activity are also covered by the temm "hirudin". Such fragments and derivatives are, for example, C-termlnally shortened desulfatohirudins.
Since not correct folded (scrambled) species and correct folded (native~ protein exist in equilibrium under denaturantt one may design conditions to tip the equilibrium in favor of the native ~;u,,lu,, 1. One possible approach is to remove the native species continuously during the folding. Another strategy is simply to recyc!e the scrambled species. This can be achieved by isolating the scrambled species and allowing them to re-equilibrate in the denaturant in order to generate the native proteln. Knowing the equilibrium constant under selected denaturing condltlons, one can calculate the total yield of native protein using the following fommula, Yield=A.(1 +B+B2+ .., +Bn) where n represents the times of recycling, A and B represent pèl~ a of native protein and scrambled species presented in equilibrium. A and B, both are smaller than 1, can be readily derived from the equilibrium constant.
Another or additional possibility ta shift the equilibrium towards the conrect folded ~iulllu,.ll~tiull is the addition of substances that promote correct folding like metal salts that stabilize the correct cu"lo", , or removing the native species using solid bound ligand that binds specifically to the native structure.
WO ~6/0342~ P~ r, ~ o ~ ' ~
In a preferred ~"~bodi"~e"l of the invention, the correct folded and the not correct folded protein are separated continuously or discontinuously.
., All denaturants that allow the solubilized protein to establish an equilibrium between not correct folded and correct folded uu,,lu,,,,dliun can be used in the process according to the invention.
The cunc~ " I of guanidine hydluulllullve is prêferably from 4 to 6 M and the cul~ ldliUII of urea is preferably 7 to 9 M.
tf the protein to be folded comprises in the correct folded l.u~lfvlllldliun one or more disulfide bonds the addition of a reducing agent or a redox system is lecu~lllllelldéd~
Advantageouslyt the process is carried out in presence of a reducing agent with a ledvx~olelllidl from -û.20 to -0.30 such as glutathione, cysteine or ~-lllell~d~ulu~l:lanul which is preferred. The ~ullcelllldliun of the reducing agent is preferably from 0.05 to 1 mM and more preferably from û.1 to û.5 mM.
The denaturation buffer or the buffer to that the correct folded protein is isolated may also contain additional compound that promote folding or prevent undesired side reactions.
Examples are further denaturants like SDS and Triton' or metal ions, further reducing agents, oxidizing agents, I..'UIII~ 19 agents like EDTA or co-enzymes.
The correct folded protein may be separated from the not corrected folded protein by any process that is able to distinguish said two forms. Said processes are, e.g., based on a difference in mobility, shape, reactivity or binding properties. Examples for suitable processes are antibody based, membrane based, ele~ upllulelic or ~,hlull.~ /yld~Jlli~.
~e~udldtion~ like gel è~ u~uhore~ gel filtration, thin layer ulllullldluyld,uhy (TLC), HPLC, affinity ulllullldluyld~lly or separation via a selective membrane. In a preferred ~ buvilllel-l of the invention the correct folded protein is separated by HPLC, TLC or affinity chromatography.
W096~03425 rcrrerg~z7z~ --h ~ 6 - 2 i 9 ~ 1, 7 In case the correct folded protein is separated discontinuously the conditions that are necessary to establish said equilibrium have to be adapted again, e.g., by cun~ ,dLiu,, or dilution of the remaining solution. It is for example possible to isolate the correct and the not-correct folded protein separately, e.g. with a HPLC column, wn~lllldl~ the fraction containing the not correct folded protein and solubilize It once more under denaturating conditions. If the separaction process is carried out .,un luuu~ly the conc~S~ of the essential cu~ll,ucin~ b can be monitored, e.g. using ~,ue~ uscùlJic means~ and wontinuously adapted.
The folding reactions is carried out preferably at a temperature that promotes the e:,ldl,li~.""e"l of an equilibrium and does not irreversibly denature the proteim Therefore, the applied temperature mainly depends on the stability of the protein and the separation procedure for the corract folded protein. For example, certain proteins of Ulelill, r ,uo,yani~ll,s are stable at 60~'C and above while proteins that originate from not hl~r",u~ liuuolydnbm~ might sometimes enter irreversible ",o lifi~ti~"~ at 40~C or below.
Brief description of the drawinos In the following ~xpeli"~e"ldl part various ~,nLio li",e"l~ of the present invention are described with reference to the accompanying drawings in which:
Fig. 1 is a HPLC protocol of the hirudin core domain ~Hir i9~ in presence of 5 M guanidine hyd,uul,luri le and 0.25 mM ~ d,ulu~tl,anol.~ig. 2 is a HPLC protocoi of epidem~ial growth factor in presence of 3 M guanidine u ;lllo,;~i~ and û.25 mM f~i-ll)el~lu~Ll,d~
96/n3425 1 ~ r ~t~
.; ~7~ ~ t 94 t 77 EXPERIMENTAL PROCEDURES
Example 1: Production and isolation of the hirudin core domain (Hir' 49) Ruco"~bi~ lI desulfated hinudin was is isolated from Saccharomyces cerevisiae asdescribed in Meyhack et al. (Thromb. Res. Suppl. Vll (1987). 33). The isolated desulfatohinudin is dissolved in 50 mM ammonh",lbi.d,l,ondl~ buffer pH 8.0 at a cuncullIldIiull of 5 mglml and digested with chymotrypsin (0.25 mg/ml~ at room temperature for 16 h. The digestion is temminated by addition of trifluoroacetic acid to a final conct~l~tldlion of 0.8 ~~O and the core domain (Hirl~9) is isolated by HPLC.
Condition for HPLC:
Column: Vydac C-18 Solvent A: 0.1 ~/O trif!uoroacetic acid in water Solvent B 0.1 ~/O trifluoroacetic acid in acetonitrile Gradient: 10 % B to 48 ~/O B in 30 min at 23~C
Retention time for Hir' 49: 14.3 min Example 2: Denaturation of the hinudin core domain ~Hir'~9) The starting material for the folding t:kur~ le,,l~, fully reduced / denatured core domain of hinudin [R] is prepared by the following method:
The hirudin core domain from example1 (2 mg/ml~ fs dissolved in Tris-HCI buffer (0.5 M, pH 8.5) containing 5 M of guanidine chloride (GdmCI) and 30 mM of dilhiuth.~;'JI.
Reduction and denaturation are carried out at 23"C for 90 min. To initiate the folding, the sample is passed through a PD-10~ column (PharmaciaJ equilibrated in 0.1 M Tris-HCI
buffer (pH 8.5). Desalting takes about 1-2 min and the sample is i"""edidI~ly used in the folding ex~u~ w~b.
WO 96103425 1 ~
-8- ~1 1 94 1 77 ExamDle 3: Foldinq of hirudin in the Dresence ot ouanidine hvdrochloride The samples are diluted to a final protein ~un.e,,l,dl;ull of 1 mg/ml; containing 0.1 M Tris-HCI buffer (pH 8.5) 5 M guanidine hy ilu~hkllid~ and 0.25 mM ;3-",e,~iaplu~l,anol. After 24 h incubation at room temperature the native hinudin is separated from scrambled hirudin via HPLC.
Gondition for HPLC:
Golumn: Vydac C-18 Solvent A: 0.1 ~/O trifluoroacetic acid in water Solvent i3: 0.1 ~/O trifluoroacetic acid In acetonitrile Gradient: 14 ~/O B to 32 5h B in 50 min at 23~C
Retention time for Hir'-49: 23 min The HPLG-protocol is given in Figure 1.
From the integration of the HPLC-protocol values the amount of native hirudin is calculated to 60% ~ 5 ~/O and the K~q to 0.67 ~ 0.15.
The fraction containing the scrambled hirudin is Iyophilized and dissolved in 0.1 M Tris-HCI
buffer (pH 8.5) containing 5 M guanldlne hy.l,u~l ,iurid~ and 0.25 mM ~-m~l~dulu~ nol to a final protein co"ue"l,~liu,- of 1 mg/ml. After 24 h incubation the native hinudin is separated from scrambled hirudin via HPLC as described above. Lyo~ and renaturation is carried out for a third time as described above.
All fractions containing the native hinudin are combined and the total recovery of native hinudin sums up to 97 ~~O after three cycles.
The actlvity of the recovered hirudin is proved by the ability to inhibit human c~-thrombin from digesting Chromozym (Boehringer Mannheim). The reaction is carried at 22~C in 67 mM Tris-HCI buffer (pH 8.0) containing 133 mM NaCI and 0.13 ~/O polyethyiene g~ycol 600Q. The rate of dlgestion was followed at 405 nm for a period of 2 min. The ~ W0 96/03425 P.~ ,3,~
~ .- , ~ .....
,.", . . ~ ~ ~, ,9 ~,UnCUll[ldliUn ot substrate is 200 mM. The l:XUllCUII~ n of thrombin is adjusted in between 2.5 and 25 nM.
For further structural analysis the recovered protein is carboxymethylated with 0.2 M
iodoacetic acid in Tris-HCI buffer (û.5 M, pH 8.5) for 30 rmin and desalted through a PD-10~
column e~ with ammonium bi ,dlbondlu solution (50 mM, pH 8.0). The disulfide contents is ot~ e(l by amino acid analysis (Chang and Knecht, Anal. Biochem. (1991), 197, 52-58) and mass spe :t~ulll~tly ~Chatrenet and Chang, J. Biol. Chem. (1992), 267, 3û38-3û43).
Exam~le 4: Foldinq of hinudin in the presence of urea The renaturation is canied out as describeal in example 3 with the sole difference that 8 M
urea is used instead of 5 M guanidine hydlu~,hlu,i.l~.
From the integration of the HPLC-protocûl values the amount of native hirudin is calculated to 9û ~~O + 5 ~~O and the Keq to 0.11 + 0.06.
Atter two cycles, the total recovery of native hirudin is 99 ~/O.
Example 5: Denaturation of epidemmal arowth factor Epidermal growth factor (EGF~ is provided by Protein Institute Inc. (Broomall, USA) and is denatured as described for hirudin in example 2.
Exam~le 6: Foldina oF EGF in the presence of auanidine h~ u~;hlu~idt:
The renaturation is carried out as described in example 3 with the sole difference that EGF
is used instead of hinudin and 3 M guanidine h~/dlu~llluli ie is used instead of 5 M guanidine hy llu :I,Iunde.
W0 ~610342!;
' " ~ A 'J ~ ~,' ' 10- 2 1 9 4 1 77 t ~, The HPLC-protocol is given in Figure 2.
From the integraUon of the HPLC-protocol values the amount of native EGF is calculated to 89~/0 + 5 ~~O and the K.q to 0.12 ~ 0.07.
After two cycles, the total recovery of native EGF is 99~/0.
The disulfide content is ~ ecl as described in example3.
Process for foldlng of pro~efns like recomb~nant hfrudfn or eptdermal growth factor .
The invention relates to a process for the ~".; " 1 of a biulugi~Al'y active and correctly folded protein in the presence of a denaturing agent.
Correct folding of genetically unyi~ee~ud proteins i5 a major issue in protein science. The problem is particularly acute, e.g., in the case of disulfide-containing proteins, which often need to be denatured and refolded to become active. Disulfide fommation is an event of post-l,a, ' " ~àl ~ . It follows the pathway of protein folding and usually ûccurs faithfully in vivo. Hûwever, in the in vitro ~ I expuli,l":"l~, many prûteins are recovered in poor yields and some do not fold into native ~;UI If Ull )r at all. To overcome this problem, a number of compounds, such as the mixture of reduced I oxidized gluta-thione (Creighton, Methods Enzymol. 131 (1986), 83-106) and protein disulfide isomerase (PDI) (Morin, J. E. & Dixon, J. E. Methods Enzymol. 113 (1985), 541-547) have been routinely used to promote the fommation of disulfides. But despite their ~ r~ use, the precise Ille-,l,dnis", of their function remains undefined and their a,, " )s have been conducted in a manner of trfal and error.
A number of pl'" ~s have appeared which report refolding attempts for individualproteins produced in bacterial hosts, or which are otherwise in a denatured or non-native foml. Formation of a dimeric, biologically active human colony stimulating factor-1 ~CSF-1 ) after expression in E. coti is described in WO 88/8ûû3 and by Halenbeck et al.
Ciul~.,-l",ology, 7 (1989), 71û-715. The plu~~dul~s described involve the steps of initial ' ' " , of CSF-1 monomers isolated from inclusion bodies under reducing conditions in a chaotropic en~;.ulll~l~lll comprfsing urea or guanidine hyd~uulllulide, refolding which is achieved by stepwise dilution of the chaotropic agents, and final oxidation of the refolded molecule in the presence of a redox-system. In WO 8818849 a process for recovering ~ulllbilldlll interleukin-2 (IL-2) is disclosed, l;ha~ault~ ed in that IL-2 isolated from refractile bodies is denatured under reduc:ng condition.s with 6M guanidine hydlu~,lllulidu, the soluble IL-2 is oxidized by a controlled oxidation in the presence of Cu2+ ions, and the oxidized IL-2 refolded by reducing the uJllcelllldliull of the denaturant in the solution.
Interleukin-2 and interferon-13 have been refolded using SDS for s~-lll' , and Cu2' ions as oxidation promoters of the fully reduced protein (US-A-4572798). The process for WO g6103~2~; PCT/EI'9~/1}27~
". '~ 2 ~ 9 4 1 7 7 isolating l~")llli illdnl refractile proteins as described in US-A-4620948 involves strong denaturing agents to solubili~e the proteins, reducing conditions to facilitate correct folding and denaturant IU; Id,ellle ll in presence of air or other oxidizing agents to refomm the disulfide bonds. A method for renaturing unfolded proteins including cytochrome c, ovalbumin and trypsin inhibitor by reversibly binding of the denaturetf protein to a solid matrix and stepwise renaturing it by diluting the denaturant is disclosed in WO 8615809.
The foregoing references are merely It:jJI(~ I ' ".'C of a huge amount of literature deaiing with the refolding of non-native proteins derived from different sources. The man skiiied in the art on the other hand knows that the success of refolding e~z,i,,,u,,l~ cannot be predicted. Unsuccessful experiments are usually not reported. There is no certainty ~hat anyone of the reported refolding conditions would work at ail with a given denatured protein containing several cysteine residues and therefore, a number of i,,l,d,,,ulecular disulfide bonds, which are reguired for activity.
Guanidine hydluuilluliLid (GdmCI) and urea are the best known denaturants for unfolding and inactivating proteins. Although the r.~el,l,dnia", ol their actions remains to be fuliy understood, it is generally evident that they disrupt the non-covalent interactions which stabiii~e the native uullfvl~ t;~ . The detrimental effect of GdmCI and urea has also been illustrated during the folding of proteins, which usually leads to the fommation of inactive, scrambled species ~Haber and Anfinsen, J. i3iol. Chem. (1962), 237, 183g-1844, Weissman and Kim Science ~1991~, 253, 1386-1393). On the other hand~ denaturants are potent agents for solubilizing intractable proteins, such as immunoglobulins and membrane ~,u~ u~e~lO etc. For example, lUCUllliJilldlll proteins expressed in an G~/~er~ d cOa system often face this problem of prolein solubility. These proteins are frequently iound in insoluble inciusion bodies and require s~ 1 by strong denaturant as the essentiai step to refoid and generate the active w"I. . ~.
Surprisingiy it has now been found that in the presence of denaturant and under equilibrium conditions correct foided protein is fommed and can be isoiated therefrom directly. This finding greatly facilitate the plU;) .rdlioll of l~ulniJilla,~l protelns as these proteins can be solubilized using denatunng agents and isolated therefrom in their correct folded native ~;unloilllclLiull without removing the solubilizing denaturant. Additionally, the presence oi ~ WO 96/03425 PCTII~P95102720 3 - 2 1 9 ~ 1 7 7 high uunu~lllldliulls of denaturant usually inactivates proteases and makes an addition of protease inllibitors unrl~u~adiy.
Therefore, the current invention provides a process for the production of a correct folded protein or a salt thereofl ,.I,d,dulcri~ed in that the protein is treated with a buffer comprfsing a denaturant, and the correct folded protein is separated therefrom directly, wherein the denaturant is selected from the group consisting of guanidine l.~J~uchludde in a COllCt:ll' ' nfrom3to7Mandureainacu,)ce"t,~t;vl,from~ito10M.
The wording correct folded protein stands for a protein that is in the native culllulllldliull andJor shows a biological activity like the enzymatic activity or the binding property of the native protein.
The inventive process is applicable to any protein or protein fragment that has to be folded into a correct folded culllolll~dliull and that e~ldL,li~lles an equilibrium between a not correct and a correct folded CUIIIOIII~ ;JII in presence of the denaturant. This is usually the case for proteins that are not irreversibly denatured by the denaturant. The ability of a protein to establish said equilibrium can easily be monitored by standard methods that provide il~loi~lldliun on the folding of proteins in solution like NMR or circular dicroism.
The protein to be refolded by the inventive process rnay be from almost any source, a - special ulclludllllulll is not necessary but not excluded. For example, a lu~,ul~lbilldlll protein that is stored in the producing host in form of inclusion bodies can be refolded by simply separating the inclusion bodies from the rest of the cell debris, 5~111' ' ,9 the proteins of the inclusion bodies with denaturant and isolating the correct folded protein therefrom. In case the protein is not stored in foml of inclusion bodies it is possible to enrich the protein only to some extend using, e.g., a uluCi,uildliull step, solubilize the protein with the denaturant and isolate the pure and correct folded protein therefrom. For ~ ~u~,u~bina~l proteins or natural proteins that are not in a correct folded cu"iu", ~ after isolation it is possible to solubilize these protein after isolation in the denaturant and isolate the correct folded fraction therefrom.
WO 9~i/0342:~ rCT~ ;fO2720 4 - ~ 1 9 4 1 7 7 Examples for suitable proteins are hinudin, epiderma! growth factor, potato ~;dl~A~p~Jiiddse inhibitor (PCI) and bovine pancreatic trypsin inhibitor ~BPTI), IGF-1, C5a-antagonist, TGF-,~. Especially preferred proteins are hirudin and epidermal growth factor ~EGF).
The temm "hinudinU as used in this invention is intended to embrace all desulfatohinudin compounds described in literature or obtainable from a tldll~lu~ ed Illi~"uo~ya"l:,"~ strain containing DNA which codes for a desulfatohirudin or a derivative thereot. Such hinudins are, for example, desulfatohinudin derivatives HV1, HV2 and HV3 (PA), as well as other hinudinproteinsasdescribede.g.byM.Schar'etaLIFEBSLett.,255(1989),1û5-110)and EP-A-347376. It is to be understood that hirudin derivatives or shorter fragments having hinudin activity are also covered by the temm "hirudin". Such fragments and derivatives are, for example, C-termlnally shortened desulfatohirudins.
Since not correct folded (scrambled) species and correct folded (native~ protein exist in equilibrium under denaturantt one may design conditions to tip the equilibrium in favor of the native ~;u,,lu,, 1. One possible approach is to remove the native species continuously during the folding. Another strategy is simply to recyc!e the scrambled species. This can be achieved by isolating the scrambled species and allowing them to re-equilibrate in the denaturant in order to generate the native proteln. Knowing the equilibrium constant under selected denaturing condltlons, one can calculate the total yield of native protein using the following fommula, Yield=A.(1 +B+B2+ .., +Bn) where n represents the times of recycling, A and B represent pèl~ a of native protein and scrambled species presented in equilibrium. A and B, both are smaller than 1, can be readily derived from the equilibrium constant.
Another or additional possibility ta shift the equilibrium towards the conrect folded ~iulllu,.ll~tiull is the addition of substances that promote correct folding like metal salts that stabilize the correct cu"lo", , or removing the native species using solid bound ligand that binds specifically to the native structure.
WO ~6/0342~ P~ r, ~ o ~ ' ~
In a preferred ~"~bodi"~e"l of the invention, the correct folded and the not correct folded protein are separated continuously or discontinuously.
., All denaturants that allow the solubilized protein to establish an equilibrium between not correct folded and correct folded uu,,lu,,,,dliun can be used in the process according to the invention.
The cunc~ " I of guanidine hydluulllullve is prêferably from 4 to 6 M and the cul~ ldliUII of urea is preferably 7 to 9 M.
tf the protein to be folded comprises in the correct folded l.u~lfvlllldliun one or more disulfide bonds the addition of a reducing agent or a redox system is lecu~lllllelldéd~
Advantageouslyt the process is carried out in presence of a reducing agent with a ledvx~olelllidl from -û.20 to -0.30 such as glutathione, cysteine or ~-lllell~d~ulu~l:lanul which is preferred. The ~ullcelllldliun of the reducing agent is preferably from 0.05 to 1 mM and more preferably from û.1 to û.5 mM.
The denaturation buffer or the buffer to that the correct folded protein is isolated may also contain additional compound that promote folding or prevent undesired side reactions.
Examples are further denaturants like SDS and Triton' or metal ions, further reducing agents, oxidizing agents, I..'UIII~ 19 agents like EDTA or co-enzymes.
The correct folded protein may be separated from the not corrected folded protein by any process that is able to distinguish said two forms. Said processes are, e.g., based on a difference in mobility, shape, reactivity or binding properties. Examples for suitable processes are antibody based, membrane based, ele~ upllulelic or ~,hlull.~ /yld~Jlli~.
~e~udldtion~ like gel è~ u~uhore~ gel filtration, thin layer ulllullldluyld,uhy (TLC), HPLC, affinity ulllullldluyld~lly or separation via a selective membrane. In a preferred ~ buvilllel-l of the invention the correct folded protein is separated by HPLC, TLC or affinity chromatography.
W096~03425 rcrrerg~z7z~ --h ~ 6 - 2 i 9 ~ 1, 7 In case the correct folded protein is separated discontinuously the conditions that are necessary to establish said equilibrium have to be adapted again, e.g., by cun~ ,dLiu,, or dilution of the remaining solution. It is for example possible to isolate the correct and the not-correct folded protein separately, e.g. with a HPLC column, wn~lllldl~ the fraction containing the not correct folded protein and solubilize It once more under denaturating conditions. If the separaction process is carried out .,un luuu~ly the conc~S~ of the essential cu~ll,ucin~ b can be monitored, e.g. using ~,ue~ uscùlJic means~ and wontinuously adapted.
The folding reactions is carried out preferably at a temperature that promotes the e:,ldl,li~.""e"l of an equilibrium and does not irreversibly denature the proteim Therefore, the applied temperature mainly depends on the stability of the protein and the separation procedure for the corract folded protein. For example, certain proteins of Ulelill, r ,uo,yani~ll,s are stable at 60~'C and above while proteins that originate from not hl~r",u~ liuuolydnbm~ might sometimes enter irreversible ",o lifi~ti~"~ at 40~C or below.
Brief description of the drawinos In the following ~xpeli"~e"ldl part various ~,nLio li",e"l~ of the present invention are described with reference to the accompanying drawings in which:
Fig. 1 is a HPLC protocol of the hirudin core domain ~Hir i9~ in presence of 5 M guanidine hyd,uul,luri le and 0.25 mM ~ d,ulu~tl,anol.~ig. 2 is a HPLC protocoi of epidem~ial growth factor in presence of 3 M guanidine u ;lllo,;~i~ and û.25 mM f~i-ll)el~lu~Ll,d~
96/n3425 1 ~ r ~t~
.; ~7~ ~ t 94 t 77 EXPERIMENTAL PROCEDURES
Example 1: Production and isolation of the hirudin core domain (Hir' 49) Ruco"~bi~ lI desulfated hinudin was is isolated from Saccharomyces cerevisiae asdescribed in Meyhack et al. (Thromb. Res. Suppl. Vll (1987). 33). The isolated desulfatohinudin is dissolved in 50 mM ammonh",lbi.d,l,ondl~ buffer pH 8.0 at a cuncullIldIiull of 5 mglml and digested with chymotrypsin (0.25 mg/ml~ at room temperature for 16 h. The digestion is temminated by addition of trifluoroacetic acid to a final conct~l~tldlion of 0.8 ~~O and the core domain (Hirl~9) is isolated by HPLC.
Condition for HPLC:
Column: Vydac C-18 Solvent A: 0.1 ~/O trif!uoroacetic acid in water Solvent B 0.1 ~/O trifluoroacetic acid in acetonitrile Gradient: 10 % B to 48 ~/O B in 30 min at 23~C
Retention time for Hir' 49: 14.3 min Example 2: Denaturation of the hinudin core domain ~Hir'~9) The starting material for the folding t:kur~ le,,l~, fully reduced / denatured core domain of hinudin [R] is prepared by the following method:
The hirudin core domain from example1 (2 mg/ml~ fs dissolved in Tris-HCI buffer (0.5 M, pH 8.5) containing 5 M of guanidine chloride (GdmCI) and 30 mM of dilhiuth.~;'JI.
Reduction and denaturation are carried out at 23"C for 90 min. To initiate the folding, the sample is passed through a PD-10~ column (PharmaciaJ equilibrated in 0.1 M Tris-HCI
buffer (pH 8.5). Desalting takes about 1-2 min and the sample is i"""edidI~ly used in the folding ex~u~ w~b.
WO 96103425 1 ~
-8- ~1 1 94 1 77 ExamDle 3: Foldinq of hirudin in the Dresence ot ouanidine hvdrochloride The samples are diluted to a final protein ~un.e,,l,dl;ull of 1 mg/ml; containing 0.1 M Tris-HCI buffer (pH 8.5) 5 M guanidine hy ilu~hkllid~ and 0.25 mM ;3-",e,~iaplu~l,anol. After 24 h incubation at room temperature the native hinudin is separated from scrambled hirudin via HPLC.
Gondition for HPLC:
Golumn: Vydac C-18 Solvent A: 0.1 ~/O trifluoroacetic acid in water Solvent i3: 0.1 ~/O trifluoroacetic acid In acetonitrile Gradient: 14 ~/O B to 32 5h B in 50 min at 23~C
Retention time for Hir'-49: 23 min The HPLG-protocol is given in Figure 1.
From the integration of the HPLC-protocol values the amount of native hirudin is calculated to 60% ~ 5 ~/O and the K~q to 0.67 ~ 0.15.
The fraction containing the scrambled hirudin is Iyophilized and dissolved in 0.1 M Tris-HCI
buffer (pH 8.5) containing 5 M guanldlne hy.l,u~l ,iurid~ and 0.25 mM ~-m~l~dulu~ nol to a final protein co"ue"l,~liu,- of 1 mg/ml. After 24 h incubation the native hinudin is separated from scrambled hirudin via HPLC as described above. Lyo~ and renaturation is carried out for a third time as described above.
All fractions containing the native hinudin are combined and the total recovery of native hinudin sums up to 97 ~~O after three cycles.
The actlvity of the recovered hirudin is proved by the ability to inhibit human c~-thrombin from digesting Chromozym (Boehringer Mannheim). The reaction is carried at 22~C in 67 mM Tris-HCI buffer (pH 8.0) containing 133 mM NaCI and 0.13 ~/O polyethyiene g~ycol 600Q. The rate of dlgestion was followed at 405 nm for a period of 2 min. The ~ W0 96/03425 P.~ ,3,~
~ .- , ~ .....
,.", . . ~ ~ ~, ,9 ~,UnCUll[ldliUn ot substrate is 200 mM. The l:XUllCUII~ n of thrombin is adjusted in between 2.5 and 25 nM.
For further structural analysis the recovered protein is carboxymethylated with 0.2 M
iodoacetic acid in Tris-HCI buffer (û.5 M, pH 8.5) for 30 rmin and desalted through a PD-10~
column e~ with ammonium bi ,dlbondlu solution (50 mM, pH 8.0). The disulfide contents is ot~ e(l by amino acid analysis (Chang and Knecht, Anal. Biochem. (1991), 197, 52-58) and mass spe :t~ulll~tly ~Chatrenet and Chang, J. Biol. Chem. (1992), 267, 3û38-3û43).
Exam~le 4: Foldinq of hinudin in the presence of urea The renaturation is canied out as describeal in example 3 with the sole difference that 8 M
urea is used instead of 5 M guanidine hydlu~,hlu,i.l~.
From the integration of the HPLC-protocûl values the amount of native hirudin is calculated to 9û ~~O + 5 ~~O and the Keq to 0.11 + 0.06.
Atter two cycles, the total recovery of native hirudin is 99 ~/O.
Example 5: Denaturation of epidemmal arowth factor Epidermal growth factor (EGF~ is provided by Protein Institute Inc. (Broomall, USA) and is denatured as described for hirudin in example 2.
Exam~le 6: Foldina oF EGF in the presence of auanidine h~ u~;hlu~idt:
The renaturation is carried out as described in example 3 with the sole difference that EGF
is used instead of hinudin and 3 M guanidine h~/dlu~llluli ie is used instead of 5 M guanidine hy llu :I,Iunde.
W0 ~610342!;
' " ~ A 'J ~ ~,' ' 10- 2 1 9 4 1 77 t ~, The HPLC-protocol is given in Figure 2.
From the integraUon of the HPLC-protocol values the amount of native EGF is calculated to 89~/0 + 5 ~~O and the K.q to 0.12 ~ 0.07.
After two cycles, the total recovery of native EGF is 99~/0.
The disulfide content is ~ ecl as described in example3.
Claims (13)
1. A process for the production of a correct folded protein or a salt thereof, characterized in that the protein is treated with a buffer comprising a denaturant, and the correct folded protein is separated therefrom directly, wherein the denaturant is selected from the group consisting of guanidine hydrochloride in a concentration from 3 to 7 M and urea in a concentration from 6 to 10 M.
2. A process according to claim 1, wherein the correct folded protein is separated continuously or discontinuously.
3. A process according to claim 1, wherein the correct folded protein is separated discontinuously.
4. A process according to claim 1, wherein the protein is hirudin or epidermal growth factor.
5. A process according to claim 1, wherein the concentration of guanidine hydrochloride is from 4 to 6 M.
6. A process according to claim 1, wherein the concentration of urea is 7 to 9 M.
7. A process according to claim 1, wherein a reducing agent is present.
8. A process according to claim 7, wherein the reducing agent has a redoxpotential from -0.20 to -0.30.
9. A process according to claim 7, wherein the reducing agent is .beta.-mercaptoethanol.
10.A process according to claim 7, wherein the concentration of the reducing agent is from 0.05 to 1 mM.
11.A process according to claim 7, wherein the concentration of the reducing agent is from 0.1 to 0.5 mM.
12.A process according to claim 1, wherein the correct folded protein is separated by antibody based. membrane based, electrophoretic or chromatographic separations.
13.A process according to claim 1, wherein the correct folded protein is separated by HPLC, TLC or affinity chromatography.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP94810437 | 1994-07-25 | ||
| EP94810437.7 | 1994-07-25 |
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| CA2194177A1 true CA2194177A1 (en) | 1996-02-08 |
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| CA002194177A Abandoned CA2194177A1 (en) | 1994-07-25 | 1995-07-12 | Process for folding of proteins like recombinant hirudin or epidermal growth factor |
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| Country | Link |
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| EP (1) | EP0804461A1 (en) |
| JP (1) | JPH11505507A (en) |
| AU (1) | AU3109795A (en) |
| CA (1) | CA2194177A1 (en) |
| FI (1) | FI970230A0 (en) |
| IL (1) | IL114699A0 (en) |
| NO (1) | NO970081L (en) |
| WO (1) | WO1996003425A1 (en) |
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| JP3734634B2 (en) * | 1999-03-03 | 2006-01-11 | ヒゲタ醤油株式会社 | Protein activation method and apparatus |
| US7521479B2 (en) | 2001-04-16 | 2009-04-21 | Panacea Pharmaceuticals, Inc. | Methods of treating prion disease in mammals |
| AU2003223936A1 (en) * | 2002-05-17 | 2003-12-02 | Kobenhavns Universitet | Method for purifying denatured proteins having a desired disulfide bond configuration |
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| GR79124B (en) * | 1982-12-22 | 1984-10-02 | Genentech Inc | |
| ES8800957A1 (en) * | 1985-02-22 | 1987-12-01 | Monsanto Co | Method of somatotropin solubilization and naturation. |
| GB8927546D0 (en) * | 1989-12-06 | 1990-02-07 | Ciba Geigy | Process for the production of biologically active tgf-beta |
| WO1992014832A1 (en) * | 1991-02-26 | 1992-09-03 | Ajinomoto Co., Inc. | Processes for purifying human bcdf |
| US5290920A (en) * | 1992-04-16 | 1994-03-01 | Allelix Biopharmaceuticals Inc. | Method of purifying human epidermal growth factor |
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1995
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- 1995-07-12 WO PCT/EP1995/002720 patent/WO1996003425A1/en not_active Application Discontinuation
- 1995-07-12 EP EP95926859A patent/EP0804461A1/en not_active Withdrawn
- 1995-07-12 JP JP8505402A patent/JPH11505507A/en active Pending
- 1995-07-24 IL IL11469995A patent/IL114699A0/en unknown
- 1995-07-24 ZA ZA956137A patent/ZA956137B/en unknown
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1997
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- 1997-01-20 FI FI970230A patent/FI970230A0/en not_active Application Discontinuation
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| EP0804461A1 (en) | 1997-11-05 |
| JPH11505507A (en) | 1999-05-21 |
| ZA956137B (en) | 1996-03-07 |
| FI970230A (en) | 1997-01-20 |
| FI970230A0 (en) | 1997-01-20 |
| WO1996003425A1 (en) | 1996-02-08 |
| NO970081L (en) | 1997-01-09 |
| AU3109795A (en) | 1996-02-22 |
| IL114699A0 (en) | 1995-11-27 |
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