United States Patent (19)
`Creighton
`
`Patent Number:
`11
`(45) Date of Patent:
`
`4,977,248
`Dec. 11, 1990
`
`54 PROCESS FOR THE PRODUCTION OFA
`PROTEIN
`76 Inventor: Thomas E. Creighton, Caxton
`Cottage, Swaffham Prior,
`Cambridge, England, CB5 OHT
`(21 Appl. No.: 391,709
`(22
`Filed:
`Aug. 10, 1989
`
`(63)
`
`Related U.S. Application Data
`Continuation of Ser. No. 939,043, filed as PCT
`GB86/00188 on Apr. 1,
`1986, published as
`WO86/05809 on Oct. 9, 1986, abandoned.
`Foreign Application Priority Data
`(30)
`Mar. 29, 1985 GB United Kingdom ................. 8508340
`51 Int. Cl................................................. C07K 3/20
`52 U.S.C. .................................... 530/412; 530/413;
`530/415; 530/416; 530/417; 530/820; 530/408;
`530/409; 530/410; 435/69.1; 435/69.7
`58) Field of Search ................................ 530/412-413,
`530/415, 416,417, 820, 408-410; 435/69.1, 69.7
`References Cited
`U.S. PATENT DOCUMENTS
`4,568,488 2/1986 Lee-Huang ........................... 424/85
`4,572,798 2/1986 Koths et al. ......
`... 530/351
`4,656,255 4/1987 Seely .............
`... 530/412
`4,659,568 4/1987 Heilman .............................. 530/417
`
`56
`
`
`
`4,766,205 8/1988 Rausch ................................ 530/412
`4,766,224 8/1988 Ghosh-Dastidar
`... 530/412
`4,839,419 6/1989 Kraemer et al. .................... 530/412
`FOREIGN PATENT DOCUMENTS
`0114506 8/1984 European Pat. Off. .
`OTHER PUBLICATIONS
`Orsini et al, JBC 253, 1978, pp. 3453-3458.
`Odorzynski et al., JBC 254, 1979, pp. 4291-4295.
`Light, Bio Techniques, vol. 3(4), 1985, pp. 298-306.
`Sofer et al, Bio Techniques, 1983 (Nov./Dec.), pp.
`198-203.
`Primary Examiner-Garnette Draper
`Attorney, Agent, or Firm-Cushman, Darby & Cushman
`57
`ABSTRACT
`A method for the renaturation of unfolded proteins
`comprises reversibly immobilizing the denatured pro
`tein on a solid phase and inducing folding of the immo
`bilized protein by progressively reducing with time the
`concentration of a denaturing agent in the solvent in
`contact with the solid phase. The refolded protein is
`recovered from the solid phase in native form. The
`proteins can be folded and recovered in high yield in a
`small volume of buffer.
`
`12 Claims, 6 Drawing Sheets
`
`Unfolded
`Cytochrome c
`
`OM
`8M
`Hor
`( Ured)
`
`Native
`Cytochromec
`
`-OM
`OM
`(NoC)
`
`I
`E
`C
`C
`O
`N
`g
`5
`2
`3
`o
`a.
`O
`
`KASHIV EXHIBIT 1069
`IPR2019-00791
`
`Page 1
`
`

`

`U.S. Patent
`
`Dec. 11, 1990
`
`Sheet 1 of 6
`
`4,977,248
`
`(IO ON )
`
`|ODN
`WO
`
`uuO82 eouDQuoSqw
`
`a
`
`|
`
`up
`O
`
`O
`
`(IOON)
`
`|
`
`WOW8 |
`( pºun) | •——————H
`
`/ 9/-/
`
`- uuO82 epubquoSqvo
`
`
`
`
`
`Page 2
`
`

`

`US. Patent
`
`Dec. 11, 1990
`
`Sheet 2 of6
`
`4,977,248
`
`‘
`
`FIG. 3A
`
`'_____—_——-—-—-—. |—.———-————’
`8* OM Urea, ImM RSSR O-O-6M NaCl, lmM RSSR,
`O-lmM RSH
`
`
`
`FIG. 3B '
`
`I
`
`Reduced
`0-! E
`
`BPTI
`g
`1
`E
`m
`.‘2’
`‘5
`a.
`
`' ....OOO'.....000: ' O 5
`O
`.____.—————-D- |————————.
`8*OM Ureo,|mM RSSR, O’O'GM NaCl, lmM RSSR,
`O-ImM RSH
`O‘lmM RSH
`|
`
`o
`E
`:
`2
`L:
`
`
`
`FIG. 3C
`
`FIG 30
`
`~
`
`iE
`
`’—
`
`E.c
`
`o
`2 ,
`E
`.25.
`f3
`V
`
`O
`
`I
`
`0
`
`gggtfied
`‘
`
`
`J
`
`l
`
`|—————-—-’
`8*OM Urea, l->OmM RSSR, 0*0'6M NGCI
`O->ImM RSH
`
`I
`
`g
`0
`Q)
`N
`8
`E
`g
`D
`< 0
`
`Reduced
`BPTl
`*
`
`
`
`g
`O-I E
`C:
`E
`m
`2
`o 2
`..0000 a .
`V
`p———-——-—-
`00.00000
`ImM RSSR,O'|mM RSH O-*O-6M NdCl,
`lmM RSSR
`
`I
`
`Page 3
`
`Page 3
`
`

`

`US. Patent
`
`Dec. 11, 1990
`
`Sheet 3 of 6
`
`4,977,248
`
`
`
`.002s:.6.meEm
`
`mmOmmsONmoOwmmOmm¢35.}mmOmmmONm.0.m0
`
`Om
`
`ON
`
`0.
`
`.002.20<mm32000_GSt
`
`Z_mOs_>IoOmn.
`
`Page 4
`
`Page 4
`
`
`

`

`U.S. Patent Dec. 11, 1990
`
`Sheet 4 of 6
`
`4,977,248
`
`Kd
`9A. - .
`67
`
`GEL
`O
`
`M 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 1819 M
`F. G. 6
`
`
`
`Kd
`94.
`67
`
`3O
`
`20
`
`1AA
`
`5.
`
`-- TIMP
`
`M 2 3 4 5 6 7 8 91O 11 12 13 14, 15 16 17 1819 M
`F. G. 7
`
`Page 5
`
`

`

`U.S. Patent Dec. 11, 1990
`US. Patent
`Dec. 11, 1990
`
`Sheet 5 of 6
`. Sheet 5 of 6
`
`4,977,248
`4,977,248
`
`
`
`Z3a
`5
`o
`2
`
`Z .
`
`4045505560657O7580
`
`5.
`
`
`
`
`
`0MNaCl
`
`OMUREA
`
`
`
`FIG.8
`
`
`
`8MUREA
`
`
`so
`
`S. 9 O
`20
`
`0
`
`
`
`3
`I00
`
`90
`
`80
`
`S 8
`so
`70
`
`so
`
`S.
`40
`
`S.
`3o
`
`Page 6
`
`Page 6
`
`

`

`U.S. Patent
`US. Patenf
`
`Dec. 11, 1990
`Dec. 11, 1990
`
`Sheet 6 of 6
`Sheet 6 of 6
`
`4,977,248
`4,977,248
`
`m6t
`
`.00220<um32000_
`
`2.
`
`1%ca
`
`O6
`
`
`
`502s:
`
`OmmuON.mmOm
`
`0vmmOm
`
`<mm:EmOL9
`
`O8
`om
`
`O/.
`ON
`
`O9
`Ow
`
`OG
`Om
`
`ow
`
`Om
`
`O2
`ON
`
`Page 7
`
`Page 7
`
`

`

`1.
`PROCESS FOR THE PRODUCTION OF A
`PROTEIN
`This is a continuation of application Ser. No.
`06/939,043 filed as PCT GB86/00188 on Apr. 1, 1986, 5
`published as WO86/05809 on Oct. 9, 1986 which was
`abandoned upon the filing hereof.
`This invention relates to processes for the production
`of proteins, in particular to the production of soluble
`native proteins. Especially the invention relates to a 10
`process for the production of a soluble native protein, in
`which an insoluble form of the protein is produced by
`host cells transformed with a vector including a gene
`coding for the protein, and as such relates to the field of
`protein production using recombinant DNA biotech- 15
`nology.
`A protein exists as a chain of amino acids linked by
`peptide bonds. In the normal biologically active form of
`a protein (hereinafter referred to as the native form) the
`chain is folded into a thermodynamically preferred 20
`three dimensional structure, the conformation of which
`is maintained by relatively weak interatomic forces such
`as hydrogen bonding, hydrophobic interactions and
`charge interactions. Covalent bonds between sulphur
`atoms may form intramolecular disulphide bridges in 25
`the polypeptide chain, as well as intermolecular disul
`phide bridges between separate polypeptide chains of
`multisubunit proteins, e.g. insulin.
`There are now numerous examples of commercially
`valuable proteins which may be produced in large quan- 30
`tities by culturing host cells capable of expressing heter
`ologous genetic material, i.e. using the techniques of
`recombinant DNA biotechnology. However in some
`cases, in particular when proteins are produced in E.
`coli host cells, the proteins are produced within the host 35
`cells in the form of insoluble protein aggregates and in
`this form do not exhibit the functional activity of their
`natural counterparts and are therefore in general of
`little use as commercial products. The lack of functional
`activity may be due to a number of factors but it is likely 40
`that such proteins produced by transformed cells are
`formed in a conformation which differs from that of
`their native form. They may also possess unwanted .
`intermolecular disulphide bonds not required for func
`tional activity of the native protein in addition to intra- 45
`molecular disulphide bonds. The altered three dimen
`sional structures of such proteins not only leads to insol
`ubility but also diminishes or abolishes the biological
`activity of the protein.
`In order to produce such proteins in a native, biologi
`50
`cally active form the insoluble protein aggregates have
`been solubilised with denaturants. The resultant solu
`tion containing the denatured protein with the individ
`ual polypeptide chains unfolded is then treated to re
`move the denaturant or otherwise reverse the denatur- 55
`ing conditions and thereby permit renaturation of the
`protein and folding of the polypeptide chains in solution
`to give protein in native, biologically active form. Pub
`lished International Patent Application No. WO
`83/04418 describes such a procedure for the production
`60
`of chymosin precursor proteins in a form capable of
`being converted to active chymosin.
`However, the usual insolubility under folding condi
`tions of fully-or partially-unfolded proteins requires
`that folding be carried out in very dilute solutions, and 65
`in large volumes. The handling of such dilute solutions
`and large volumes can add significantly to the cost
`when such processes are applied industrially.
`
`4,977,248
`2
`I have now found that unfolded proteins may be
`adsorbed reversibly on solid phase adsorbents, induced
`to fold whilst bound to the adsorbent by progressively
`reducing with time the concentration of denaturing
`agent in the solvent in contact with the adsorbent, and
`then eluted from the adsorbent to yield the protein in
`native form. This provides a process for the production
`of soluble native proteins which avoids the requirement
`for refolding of the protein in very dilute solution, and
`in large volumes, and thus provides benefits for indus
`trial scale applications. The process further provides a
`way of separating different proteins or different confor
`mational states. Other advantages and benefits of this
`process are described hereinafter and will be apparent
`to those skilled in the art of protein production. The
`process may be applied advantageously to proteins pro
`duced by recombinant DNA biotechnology which are
`produced within host cells in the form of insoluble pro
`tein aggregates.
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a graph showing the refolding of horse
`cytochrome c adsorbed to CM-cellulose.
`FIG. 2 is a graph showing the refolding of hen oval
`bumin adsorbed to DEAE-cellulose.
`FIG. 3 is a graph showing the refolding of reduced
`bovine pancreatic trypsin inhibitor (BPTI) adsorbed to
`CM-cellulose.
`FIG. 5 is a graph showing the elution profile from a
`column carrying adsorbed prochymosin.
`FIGS. 6 and 7 are reproductions of SDS-polyacryla
`mide gels showing fractions eluted from columns
`loaded with prochymosin, tissue inhibitor of metallo
`proteinases (TIMP) and porcine growth hormone
`(pGH).
`FIG. 8 is a graph showing the elution profile from a
`Pharmacia Mono QFTLC column loaded with TIMP,
`monitored at 280 nm.
`FIG. 9 is a graph showing the elution profile from a
`Pharmacia Mono QFTLC column loaded with porcine
`growth hormone monitored at 280 nm.
`Accordingly the present invention comprises a pro
`cess for the production of a protein in native form, in
`which a solution of the denatured protein is contacted
`with a solid phase which reversibly binds the protein
`and the denatured protein is reversibly bound to the
`solid phase, characterised in that, the bound protein is
`renatured by progressively reducing with time the con
`centration of the denaturing agent in the solvent in
`contact with the solid phase, and the renatured protein
`is recovered from the solid phase in native form.
`The process of the invention may be applied to any
`protein which is produced in a non-native form e.g. in
`the form of insoluble protein aggregates. The process
`may be used to convert the non-native form of the pro
`tein to the native form, e.g. the soluble native form of
`the protein. In particular the invention may be applied
`to proteins produced by recombinant DNA biotechnol
`ogy, in which an insoluble form of the protein is pro
`duced by host cells transformed with a vector including
`a gene coding for the protein. Examples of "recombi
`nant proteins produced in insoluble form by trans
`formed host cells are porcine growth hormone, tissue
`inhibitor of metalloproteinases (TIMP), insoluble chy
`mosin precursors e.g. met-prochymosin, immunoglobu
`lins and CAT-fusion proteins e.g. CAT-calcitonin, pro
`duced in bacteria (e.g. E. coli) or yeast (e.g. S. cerevisiae)
`host cells.
`
`Page 8
`
`

`

`4.
`MATERIALS AND METHODS
`
`15
`
`Proteins
`Horse cytochrome c was obtained from BDH. Hen
`ovalbumin was obtained from Sigma. Bovine pancreatic
`trypsin inhibitor (BPTI, R Trasylol) was the generous
`gift of Bayer AG.
`Ion Exchange Resins
`The carboxymethyl-(CM) and diethylaminoethyl
`(DEAE) cellulose resins were respectively the CM52
`and DE52 products of Whatman.
`Unfolding of Proteins
`Proteins were unfolded by dissolving the dry native
`protein obtained commcercially to a concentration of 5
`to 10 mg/ml in 8M urea also containing the appropriate
`buffer. Unfolding of ovalbumin was ensured by heating
`the solutions at 50 for 15 minutes. When disulphides
`were also to be reduced, dithiothreitol was included at
`50 mM. Urea was the Aristar grade of BDH, and all
`urea solutions were prepared just prior to use.
`Solid-state refolding of proteins
`Small columns of the appropriate resin (generally 1.5
`cm in diameter and 3 cm in length) were equilibrated
`with the desired buffer in which the protein was deter
`mined to adsorb tightly. Most frequently, this was 10 to
`100 mM Tris-HCl at pH 8.7 with 1 mM EDTA. Just
`before applying the unfolded protein, the column was
`washed with a few ml of the same buffer solution con
`taining 8M urea. Usually 10 to 20 mg of unfolded pro
`tein in 2 ml of 8M urea was applied to the column. It
`was followed by the buffers designed to induce the
`adsorbed protein to fold, usually using a linear gradient
`to remove gradually the urea and to vary other parame
`ters. The protein was then eluted with an appropriate
`salt gradient and the elution profile compared with that
`of the same amount of protein that had not been un
`folded. The flow rates varied between 1.0 and 3.3
`ml/min.
`The absorbance at 280 nm of the eluate was moni
`tored continuously, and fractions of 200 drops (about 13
`ml) collected. Thiols were assayed by the method of
`Ellman (Arch. Biochem. Biophys 82:70 (1959)) and
`active BPTI by its ability to inhibit trypsin (Kassell B.,
`Methods Enzymol 19, 844 (1970)). Assays were per
`formed at 25, all other manipulations were at room
`temperature.
`
`4,977,248
`3
`The protein may be denatured in any suitable dena
`turant solution. The solution of the denaturing agent
`may be chosen having regard to the solid phase to be
`used for reversible binding of the denatured protein.
`The denaturing agent may be in aqueous solution, such
`as for example an aqueous solution of guanidine hydro
`chloride, though is preferably urea, e.g. 8M urea.
`The solid phase may be any solid phase to which the
`denatured protein may be reversibly bound. The pro
`tein may be bound to the solid phase by reversible cova
`10
`lent linkage. Alternatively the solid phase may comprise
`an adsorbent for the protein. For instance the solid
`phase may be an ion-exchange resin such as an agarose
`or similar material e.g. Q-sepharose or S-sepharose,
`Pharmacia Mono Q
`FPLC, Pharmacia Mono S FPLC, or cellulose, e.g.
`CM-cellulose, DEAE-cellulose, phospho-cellulose, or
`Amberlite of which CM-cellulose and Pharmacia Mono
`QFPLC are preferred. It will be appreciated that when
`the solid phase is an ion-exchange resin the denaturant
`solution used is typically of low ionic strength to pro
`mote adsorption of the protein.
`The solid phase may comprise a continuous solid
`phase such as a surface, though is preferably particulate.
`25
`Advantageously the solid phase may be in the form of a
`column containing particles of the solid phase through
`which the denaturant solution and solvent may be
`flowed.
`Renaturing of the protein bound to the solid phase is
`30
`effected by progressively reducing with time the con
`centration of denaturing agent in the solvent in contact
`with solid phase to which the protein is bound. In a
`preferred embodiment the protein bound to the solid
`phase is treated with a solvent gradient, preferably a
`35
`linear gradient, in which the composition varies from
`one in which the solvent initially presents a denaturing
`environment to one in which the solvent presents a
`natural environment to the protein. For example a urea
`gradient varying from an initial urea concentration of 0
`8M to a final concentration of OM may be used. The
`composition of the gradient is typically varied in a con
`tinuous manner, e.g. linearly, over a prolonged period
`of time, usually at least over about 30 minutes and pref.
`erably over about 60 minutes. Preferably the solvent
`45
`gradient is passed through a column containing bound
`protein.
`In addition to denaturant the solvent which is con
`tacted with the solid phase may contain other compo
`50
`nents such as cofactors, disulphide forming and break
`ing reagents and salts. The concentrations of these other
`components may be varied as desired during renatur
`ation of the protein.
`On completion of renaturation treatment the protein
`is recovered from the solid phase. For instance rena
`tured protein may be recovered from an ion-exchange
`resin column by elution with a salt, e.g. NaCl, gradient.
`The invention also extends to a protein whenever
`produced by the process according to the invention.
`60
`The invention is further described by way of illustra
`tion only in the following examples. It will be appreci
`ated that the results presented in the examples demon
`strate the feasibility of the process of the invention for
`production of proteins in general in native form.
`EXAMPLE 1.
`This Example relates to studies of model proteins.
`
`55
`
`a. Horse Cytochrome c
`Horse cytochrome c (FIG. 1) was unfolded in 8M
`urea (Myer Y. P., Biochemistry 7 765 (1968); Stellwa
`gen E., Biochemistry 7 289 (1968)) and applied to a
`small column of CM-cellulose at low ionic strength,
`where it was observed from its colour to bind to the top
`1 to 2 mm of resin. The urea concentration of the sol
`vent was gradually lowered by a linear gradient of 8M
`to OM urea. The adsorbed protein was then quantita
`tively eluted with a gradient of increasing salt concen
`tration. No coloured protein was retained by the col
`umn and the elution profile was indistinguishable from
`that of cytochrome c that had not been unfolded. The
`same result was obtained if the unfolded cytochrome c
`was dispersed by stirring it into the resin of the top third
`of the column.
`If the urea was not removed gradually with a gradi
`ent, but omitted entirely from buffer applied to the
`column after the unfolded protein, only approximately
`80% of the protein was eluted; the remainder was re
`
`65
`
`Page 9
`
`

`

`15
`
`4,977,248
`6
`5
`tained at the top of the column. Further experimental
`There was little dependence of the product on the
`details are described below in the legend to FIG. 1.
`type of RSH/RSSR gradient (FIG. 3b, c). However, it
`FIG.1. Refolding of horse cytochrome c adsorbed to
`was necessary to have a gradient of decreasing urea
`CM-cellulose. The buffer throughout was 0.1M Tris
`concentration; an abrupt removal of the urea resulted in
`HCl, pH 8.7, 1 mM EDTA. 10.0 mg of cytochromec
`recovery of only a small fraction of the BPTI, and the
`dissolved in 1.0 ml of buffer (native, or unfolded with
`inactive form predominated (FIG. 3d). Significantly
`8M urea also present) was applied to a column of CM
`higher yields of correctly folded BPTI were generated
`cellulose equilibrated with the same buffer. The un
`using a higher ionic strength buffer throughout (FIG.
`folded protein was followed by a 200-ml linear gradient
`4), where the protein would be expected to be less
`of 8M to OMurea in buffer. Both proteins were eluted
`tightly adsorbed to the resin.
`10
`with a 200 ml linear gradient of OM to 1.OMNaCl. The
`Further experimental details are provided in the leg
`elution profile shown is given by the absorbance at 280
`ends to FIGS. 3 and 4.
`FIG. 3. Refolding of reduced BPTI adsorbed to CM
`cellulose. In each case, 20 mg of BPTI in 2 ml of 8M
`urea plus buffer was adsorbed. A is the control in which
`the BPTI was not reduced, but only dissolved in 8M
`urea, where it remains folded and the disulphides intact;
`B, C and D used reduced BPTI by inclusion of 50 mM
`dithiothreitol, which is the absorbing material not
`bound to the column. In A, B, and C, the protein was
`followed by a 200 ml linear gradient of 8M to OMurea
`in buffer; in D there was no urea in the subsequent
`buffer and no gradient. 1.0 mM hydroxyethyl disulfide
`(RSSR) was present uniformly in the gradient buffers in
`A, B, and D; in C it was present only in the 8M urea
`solution and consequently decreased in concentration.
`Mercaptoethanol (RSH) was present uniformly at 0.1
`mM in B and D, but in C was present at 1.0 mM in the
`OM urea solution only and consequently increased in
`concentration. The proteins were eluted with 200 ml
`linear gradients of OM to 0.6MNaCl in buffer. In A and
`B these solutions also contained 1.0 mM RSSR and 0.1
`mM RSH; in D only 1 mM RSSR.
`The buffer throughout was 10 mM Tris (pH 8.7) and
`1 mM EDTA. The solid line gives the absorbance at 280
`nm, which monitors both RSSR and protein.
`FIG. 4. Improved yield of correctly refolded BPTI
`using 0.1M Tris-HCl, pH 8.7, 1 mM EDTA buffer
`throughout. The conditions were as in FIG. 3c, except
`for the ten-fold higher Tris concentration; also 1.0 mM
`RSSR was present uniformly throughout the urea gra
`dient, whereas the concentration of RSH increased
`linearly from OM to 1.0 mM.
`Protein not eluted from the column may be recovered
`by going through a second treatment involving 8M urea
`and 50 mM dithiothreitol, followed by the refolding and
`disulphide formation steps.
`A wide variety of solid supports may be used. Vari
`ous hydrophilic ion-exchange resins were used here
`because of their availability and widespread use in chro
`matography of proteins, but there are very many other
`chromatographic supports that might be suitable. The
`electrostatic binding of the protein to the resin is not
`expected to interfere greatly with folding, since modifi
`cation of the ionized groups of at least some proteins
`does not alter substantially their folding or stability.
`Globular proteins generally have all ionic groups on
`their surface. Non polar supports would be expected to
`interfere with the hydrophobic interaction that is im
`portant for folding of globular proteins, but might be
`ideal for membrane proteins.
`The solvents used must be compatible with both ad
`sorption of the protein to the solid support and variation
`of the folding conditions. Other parameters beside dena
`turant concentrations may be used to vary the folding
`conditions, such as temperature, pH, ionic strength,
`disulphide formation or breakage and ligand concentra
`
`b. Ovalbumin
`Unfolded ovalbumin is well-known to precipitate to a
`large extent upon removal of denaturant (Simpson etal,
`J. Amer. Chem. Soc. 75 5139 (1953)). The protein is
`usually heterogeneous, due to partial phosphorylation
`of two residues and the proteolytic cleavage, and the
`20
`major doubly-phosphorylated species has been shown
`to unfold reversibly (Ahmad and Salahuddin, Biochem
`istry 1551.68 (1976)). When heterogeneous commercial
`ovalbumin was refolded while adsorbed to DEAE-cel
`lulose, using a procedure like that employed with cyto
`25
`chrome c, a yield of approximately 50% of apparently
`refolded molecules was obtained (FIG. 2). Further ex
`perimental details are described below in the legend to
`FIG. 2.
`FIG. 2. Refolding of hen ovalbumin adsorbed to
`30
`DEAE-cellulose. The buffer throughout was 20 mM
`Tris-HCl, pH 8.7.20 mg of ovalbumin dissolved in 2.0
`ml of buffer (native, or unfolded with 8M urea also
`present) was applied to a column of DEAE-cellulose
`equilibrated with the same buffer. The unfolded protein
`35
`was followed by a 200 ml linear gradient of 8M to OM
`urea. Both proteins were eluted with a 200 ml linear
`gradient of OM to 0.5M. NaCl.
`c. Bovine pancreatic trypsin inhibitor
`Some proteins must form disulphides to attain a stable
`folded conformation, and this permits further manipula
`tion and elucidation of the folding process. The protein
`best characterised in this respect is BPTI, with three
`disulphide bonds. Reduced BPTI protein in 8M urea
`45
`and 50 mM dithiothreitol was applied to CM-cellulose
`columns; the urea concentration was gradually dimin
`ished with a linear gradient coupled to various alter
`ations of the thiol/disulphide redox potential via the
`concentrations of the thiol and disulphide forms of glu
`50
`tathione, mercaptoethanol, or cysteamine, respectively
`negatively charged, neutral and positively charged. The
`protein was then eluted with a salt gradient (FIG. 3).
`The absorbance at 280 nm detected both protein and
`the disulphide reagent; thiols and BPTI trypsin inhibitor
`55
`activity were assayed in the eluant. These procedures
`measured the reactivity of the thiols with the disulphide
`reagent (to generate reduced reagent) and the propor
`tion of eluted protein that had a native-like conforma
`60
`tion.
`At least 90% of the BPTI could be eluted from the
`column after removing the urea from the buffer by a
`linear gradient, which also included thiol and disulfide
`forms of mercaptoethanol, RSH and RSSR respec
`tively, in constant, increasing, or decreasing concentra
`65
`tions. The disulphide reagent was included to generate
`protein disulphides, the thiol reagent to assist in protein
`disulphide rearrangements.
`
`Page 10
`
`

`

`4,977,248
`7
`tions. Specific cofactors may also be included in the
`solvent to vary the folding conditions.
`The unfolded protein adsorbed to the ion-exchange
`resin is accessible to at least small reagents, as shown by
`the reactivity of the thiol groups of BPTI. Disulfide
`bond formation is one type of post-translational modifi
`cation, so others might also be possible, such as glycosy
`lation, y-carboxylation and hydroxylations. This would
`require that the adsorbed protein be accessible to the
`necessary reagents and enzymes.
`A procedure to minimize aggregation of the protein
`bound to the solid support may be to use one or a few
`covalent, but reversible, attachments of the protein to a
`solid support.
`
`8
`-continued
`FIG. 6
`
`Sample
`Fraction 44
`Fraction 45
`Fraction 6
`Fraction 7
`Pharmacia Markers
`
`FPLC run - Chymosin
`
`Track No.
`6
`7
`18
`19
`20
`
`O
`
`15
`
`EXAMPLE 2
`Prochymosin
`The cells used in this experiment were E. coli HB101
`cells carrying the plasmid pCT70 which contains the
`20
`gene coding for prochymosin. A full description of this
`plasmid and its derivation is provided in British Patent
`No. 2100737B.
`10 g wet weight cells (HB101/pCT70) were sus
`pended in 30 ml 50 mM TrisCl pH 8.0, 1 mM EDTA, 50
`25
`mMNaCl and lysed by one pass through a French press
`at 1250 p.s. i. The lysate was centrifuged at 12000 Xg for
`5 minutes at 4 C. The pellet was suspended in 30 ml
`H2O and centrifuged as before. The washed pellet was
`then suspended in 8 ml 20 mM TrisCl pH 9.0, 1 mM
`EDTA, 8Murea and incubated for 30 minutes at 30' C.
`A 10 mm x 100 mm Pharmacia Mono QFPLC col
`umn was equilibrated with 20 mM ethanolamine pH 9,
`8M urea. 200 ul of the prochymosin urea suspension
`was applied to the column at a flow rate of 0.5 ml.
`35
`min. A linear decreasing gradient of 8M to OM urea
`in 20 mM ethanolamine pH 9.0 was applied to the col
`umn at a flow rate of 0.5 ml.min-1 over a total period of
`60 minutes. 1 ml fractions were collected. The column
`was then washed with 10 ml 20 mMethanolamine pH
`40
`9.0 and developed with a linear gradient of OM-1M
`NaCl in 20 mMethanolamine pH 9.0 at a flow rate of
`0.5 ml.min-1 over a total period of 60 minutes. 1 ml
`fractions were collected, FIG. 5 shows the elution pro
`file from this column, monitored at 280 nm.
`45
`Fractions collected from the column, as described
`below in the legends to FIGS. 6 and 7 were analysed by
`SDS Page on a 12.5% acrylamide gel run at 150 V for
`4 hr according to the method of Laemmli (Nature 227
`680-685 (1970)). The gel was stained with coomassie
`50
`blue, destained in 7.5% acetic acid and then stained with
`silver stain (Merril et al, Science 211 1437 (1981)).
`
`2
`3
`4
`5
`6
`7
`8
`9
`
`FIG. 6
`
`Sample
`Pharmacia Markers
`Fraction 66
`Fraction 67
`Fraction 68
`Fraction 70
`Fraction 71
`Fraction 72
`Fraction 73
`Fraction 74
`Fraction 75
`Fraction 76
`Fraction 77
`Fraction 34
`Fraction 37
`Fraction 43
`
`FPLC run - pCH
`
`FPLC run - TIMP
`
`55
`
`65
`
`FPLC run - TIMP
`
`r
`FPLC run- Chymosin
`
`FPLC run - Chymosin
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`
`FIG. 7
`
`Sample
`Pharmacia Markers
`Fraction 74
`Fraction 75
`Fraction 76
`Fraction 77
`Fraction 78
`Fraction 40
`Fraction 41
`Fraction 43
`Fraction 44
`Fraction 62
`Fraction 63
`Fraction 64
`Fraction 74
`Fraction 75
`Fraction 76
`Fraction 77
`Fraction 78
`Fraction 79
`Pharmacia Markers
`
`EXAMPLE 3
`Tissue Inhibitor of Metalloproteinases (TIMP)
`The cells used in this experiment were E. coli E103S
`cells carrying the plasmid pMG461 which contains the
`gene coding for TIMP. The derivation of plasmid
`pMG461 is described in our co-pending British patent
`application No. 8600199 filed Jan. 6, 1986.
`The experiment was performed essentially as de
`scribed in Example 2, using E. coli E103S cells carrying
`plasmid pMG461 in place of E. coli HB101 carrying
`plasmid pCT 70.
`FIG. 8 shows the elution profile from the Pharmacia
`Mono QFPLC column monitored at 280 nm.
`Fractions collected from the column as described
`above in the legends to FIGS. 6 and 7 were analysed as
`described in Example 2.
`EXAMPLE 4
`Porcine Growth Hormone
`The cells used in this experiment were E. coli E1035S
`carrying the plasmid pMG935. A gene coding for por
`cine growth hormone may be derived as described in
`published European patent applications Nos. EP
`104920A (Biogen) and EP 111389A (Genentech).
`Plasmid pMG935 is a derivative of pMG196, carry
`ing the gene encoding porcine growth hormone.
`pMG 196 is a dual-origin expression vector carrying the
`pTrp promoter and T7 terminator. To construct
`pMG196 a XhoI-BamHl DNA fragment carrying the
`Col El origin from pMG15 was ligated to an EcoR1
`BamHl DNA fragment isolated from pMG411 (Yarran
`ton et al Gene 28293-300 (1984)), and to an EcoR1-SalI
`DNA fragment carrying ApR and cI857 from pCOV2
`(Queen J. Mol. and Applied Genet. 2 1-10 (1983))
`which had a Sall-HindIII DNA linker inserted at the
`
`Page 11
`
`

`

`4,977,248
`10
`9
`2. A process according to claim 1 where the solid
`unique BamHl site of this plasmid. The resulting plas
`phase is treated with a solvent gradient in which the
`mid was cleaved with EcoRI and Pst and the DNA
`composition varies from one in which the solvent ini
`fragment carrying the pSC101 origin replaced with an
`tially presents a denaturing environment to one in
`EcoRI to Pst DNA fragment from pMG168, carrying
`which the solvent presents a natural environment to the
`a pSC101 origin and par function. This plasmid is
`protein.
`pMG171. pMG171 was cleaved with BamH1 and PstI
`3. A process according to claim 2 wherein the solvent
`and the small DNA fragment replaced with a BamHl
`gradient is a linear gradient.
`Pst fragment isolated from pCT54 (Emtage etal PNAS
`4. A process according to claim 1 where the denatur
`USA 80 (1983) 3671-3675). This fragment carried the
`ing agent is urea.
`10
`pTrp and T7 terminator and the resulting plasmid is
`5. A process according to claim3 wherein the solvent
`pMG 196 (Wright et al submitted for publication). The
`gradient is a urea gradient which varies from an initial
`gene for pGH was assembled into pMG196, by inserting
`urea concentration of 8M to a final concentration of OM
`into partially EcoRI cleaved, Cla cleaved vector, and
`over a period of 60 minutes.
`after ligation and transformation amplicillin resistant
`6. A process according to claim 1 where the protein
`transformants were selected at 30 C. The plasmid iso
`is a protein produced by recombinant DNA biotechnol
`lated from these transformants is pMG935, and ex
`ogy in which an insoluble form of the protein is pro
`presses pGH from the pTrp.
`duced by host cells transformed with a vector including
`The experiment was carried out essentially as de
`a gene coding for the protein.
`scribed in Example 2, using E. coli E103S carrying
`7. A process according to claim 1 where the solvent
`20
`plasmid pMG935 in place of E. coli HB101 carrying
`which is contacted with the solid phase contains one or
`plasmid pCT 70.
`more components selected from denaturing agent, co
`FIG. 9 shows the elution profile from the Pharmacia
`factors, disulphide forming and breaking reagents and
`Mono QFPLC column monitored at 280 nm.
`salts.
`Fractions collected from the column as described
`8. A process according to claim 7 where the solvent
`25
`above in the legend to FIG. 6 were analysed as de
`which is contacted with the solid phase contains urea
`scribed in Example 2.
`and dithiothreitol.
`9. A process according to claim 1 where the solid
`I claim:
`1. A process for the production of a protein in native
`phase adsorbent is an ion-exchange resin.
`form, in which a solution of the denatured protein is
`10. A process according to claim 1 where the solid
`contacted with a solid phase and the denatured protein
`phase adsorbent is cellulose.
`is reversibly bound to the solid phase, characterised in
`11. A process according to claim 1 where the solid
`that the bound protein is renatured by progressively
`phase adsorbent is an agarose or similar material.
`12. A process according to claim 1 where the rena
`reducing with time the concentration of denaturi