CA1289492C

CA1289492C - Process for the production of chymosin

Info

Publication number: CA1289492C
Application number: CA000429825A
Authority: CA
Inventors: Norman Henry Carey; Michael Terence Doel; Timothy John Roy Harris; Peter Anthony Lowe; John Spencer Emtage
Original assignee: Celltech R&D Ltd
Current assignee: Chr Hansen AS
Priority date: 1983-03-25
Filing date: 1983-06-07
Publication date: 1991-09-24
Anticipated expiration: 2008-09-24
Also published as: GB8308234D0

Abstract

A PROCESS FOR THE PREPARATION OF CHYMOSIN
ABSTRACT

There is described a process for the preparation of chymosin in which an insoluble form of a chymosin precursor, such as methionine-chymosin, is produced by a host organism, such as Saccharomyces cerevisiae or E.coli, transformed with a vector including a gene coding for the chymosin precursor, wherein the insoluble form of the chymosin precursor is solubilised to produce a soluble native form of the chymosin precursor prior to cleaving the soluble native form of the chymosin precursor to produce chymosin. The solubilisation involves denaturing the insoluble chymosin precursor and then allowing it to renature forming the normal or native form of the chymosin precursor. The native form may then be cleaved to form chymosin. The denaturants used include aqueous solutions of urea or guanidine hydrochloride and alkaline solutions of pH between pH 9.5 and pH 11.5 either separately or in combination. In a particular type of colubilisation described, the insoluble chymosin precursor is denatured using a urea or guanidine hydrochloride solution. The resulting solution is then diluted into an alkaline solution to yield the native form of the chymosin precursor.

Description

~LZ~ 2 This invention relates to the field of protein production using recombinant DNA biotechnology. In particular it relates to a process for preparing chymosin from an insoluble form of a chymosin precursor produced by a host organism transformed with a vector including a gene coding for the chymosin precursor.
ThPre are now numerous examples of commercially valuable proteins which may be produced in large ~uantities by culturing a host organism capable of expressing heter-ologous genetic material. Once a protein has been produced by a host organism it is usually necessary to treat thehost organism in some way, in order to'obtain the desired protein in a free form. In some cases, such as in the production of the interferon in E. coli a lysis or permea-bilisation treatment alone may be sufficient to affordsatisfactory yields.' However, some protelns are pro~uced withln a h~t organism in the form of insoluble protein aggregates which are nok susceptible to extraction by lysis or permeabilisation treatment alone. It has been reported for instance that a human insulin fusion protein produced in E. coli forms insoluble protein aggretates (see D.C. Williams et al Science vol. 215 pges 687-689). In the normal bio~ogically active form ~hereinafter referred to as the native formj a protein exists as a chain of amino acids linked by peptide bonds. The chain is folded lnto-~a thermodynamically preferred three dimensional structure, the conformation of which is maintained by relatively weak interatomic forces such as hydrogen bonding, hydrophobic interactions and charge interactions.

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A number of S-S covelent bonds may form intramoleculer brid-ges in the polypeptide chain. The insoluble proteins pro-duced by certain host organisms do not exhibit the function-al activity of their naturel 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 that such proteins produced by transformed host organisms are formed in a conformation which differs from that of their native states. The altered three dimen-sional structure of such proteins not only leads to insolu-bility but also diminishes or abolishes the biological ac-tivity of the protein. It is not possible to predict whether a given protein expressed by a given host organism will be soluble or insoluble.
In our copending published British patent applica-tion GB2100737A ~which corresponds with Canadian Patent Application Serial No. 404,600), we describe a process for the production of the proteolytlc enzyme chymoein. The pro-cess involve~ cleaving one of the chymosin precursor poly-Z peptides; preprochymosin, methionine-prochymosin or methio-ninechymosin, which may be expressed rom a host organism which has been transformed with a vector including a gene coding for the relevant protein. The process for preparing a host organism transformed with a vector carrying a suit-- 25 able gene is described in detail in the pending Canadian and British patent applications.
In the course of our work relating to the process for preparing chymosin we discovered that the chymosin pre-cursor proteins produced by various host organisms used were not produced in théir native form. In particular the me-thionine-prochymosin produced by E. coli is almost entirely produced as an insoluble aggregate and about 75% of the methionine-prochymosin produced in Saccharomyces cerevisiae is produced in an insoluble form.

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In order to produce a chymosin precursor in a native form which may be cleaved to form active native chymosin the proteins produced by a host organism must be solubilised and converted into their native form before the standard techniques of protein purification and cleavage may be applied.
According to the present invention there is pro-vided a process for the production of chymosin in which an insoluble form of a chymosin precursor is produced by a host organism transformed with a vector including a gene coding for the chymosin precursor wherein the insoluble form of the chymosin precursor is solubilised to produce a soluble native form of the chymosin precursor prior to cleaving the soluble native form of the chymosin precursor to produce chymosin, the solubilisation inyolving reversibly denaturing the insoluble form of the chymosin precursor and subsequently allowing the chymosin precur~or to renature, thereby forming the soluble natlve form of the chymosin precursor.
Preferably the insoluble form of the chymosin pre-cursor produced by the host organism is preprochymosin or a ~' fusion protein including the amino acid sequénce of prepro-chymosin, prochymosin or chymosin, for example, methionine-prochymosin or methionine-chymosin. Methionine-prochymosin is preferred as the chymosin precursor. The host organism may be a host organism or the progeny of a host organism which has been transformed (using the techniques described in our published British patent application GB2100737A) with a vector including heterologous genetic material coding for the chymosin precursor. The host organism may be a yeast, for example,~ Sacchar~myce5'cerevisiae or a bacterium, for example, E. coli! B. s~btilis,~B.~st~ar~the~mophilis, or PSèudo~as, Saccha~o~ce5 cére~iSiae or E. cail are the preferred host organisms.
The expression system comprising _ caii trans-formed with a vector including a gene coding for the chymosln '~

precursor, methionine-prochymosin, is especially preferred.
The term 'insoluble' as used herein means in a form which, under substantially neutral conditions (for example pH in the range 5.5 to 8.5), is substantially insoluble or is in insolubilised association with insoluble material produced on lysis of host organism cells. The insoluble product is either produced within the cells of the host organism in the form of insoluble relatively high molecular weight aggregates or may simply be associated with insoluble cell membrane material.
In order to produce the native form of the chymosin precursor it is necessary to alter the conformation of the insoluble product produced by the host organism. This may be done by denaturing the insoluble protein. Denaturing has the ef~ect o~ abolishing the weak interatomic forces which maintain the protein in its three dimensional form causing the protein to unfold. The covalent bonds between adjacent atoms in the protein are left intact, including S-S bonds which maintain some of the three dimensional structure of the protein. The denatured state of a protein is less compact and is generally catalytically inactive. The denatured state is however usually soluble in the denaturing solution used. Removal of the denaturant from the solubilised proteins results in the refolding of the protein to produce the thermodynamically preferred native state of the protein.
The renaturing is accompanied by the appearance of biological activity.
According to a preferred aspect of the invention the solubilisation involves reversibly denaturing the insoluble form of the chymosin precursor and su~sequently allowing the chymosin precursor to renature, thereby producing the soluble native form of the chymosin precursor.
Preferably the insoluble ~orm o~ the chymosin precursor is denatured in an aqueous solution comprising . ~ , ' ~9~92 urea at a concentration of at least 7M and the chymosin precursor is renatured subsequently by reducing the concentration of urea in the solution below a concentration effective to denature the chymosin precursor, to produce a soluble native form of the chymosin precursor.
When the insoluble chymosin precursor is treated with urea the insoluble precursor is completely solubilised. The disulphide intramolecular bridges of the protein are however preserved and subsequently act as a nucleus for refolding. When the urea is removed, for example by dialysis, the protein returns to a thermodynamically stable conformation which, in the case of chymosin precursors, is a conformation capable of being converted to active chymosin by the methods described in published British patent application GB2,100,737A. The renatured protein~ have the solubility characteri~tics of the native protelns.
In a ~urther preferred aspect the insoluble form o~ the chymosin precursor is denatured in an a~eous ~0 solution comprising guanidine hydrochloride at a concentration of at least 6M and the chymosin precursor is renatured subsequently by reducing the concentration of guanidine hydrochloride in the solution below a concentration effective to denature thé chymosin precursor, to produce a soluble native form of the chymosin precursor.
The physical effect of using guanidine hydrochloride is as discussed above ~or urea.
In a further preferred aspect the insoluble ~orm ~ of the chymosin precursor is denatured in an alkaline ,~ 30 aqueous so}ution of between pH 9 and pH 11.5 and the chymosin precursor is renatured subsequently by reducing the pH of the solution below a pH effective to denature the chymosin precursor, to produce the soluble native ~orm of the chymosin precursor. Preferably the alkaline aqueou~
solution is o~ pH between pH 10 and pH 11. Most preferred :

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12~39~2 is an alkaline aqueous solution of pH from 10.5 to lo.9, preferably about 10.7.
Treatment of an insoluble chymosin precursor extract with an alkaline solution as described above does not result in complete solubilisation of the chymosin precursor. Since insoluble material such as cell debris is present at all times, a number of mass transfer effects are important. It has been found that multiple extractions with alkali are more efficient than a single extraction even when large extraction volumes are used. This also has the adva~tage of minimising the time for which the solubilised chymosin precursor is in contact with alkali.
This is of importance since there is evidence that prochymosin slowly loses activity in alkaline solutions.
Preferably, therefore, in this aspect of the invention the in~oluble form of the chymosin precursor is present in con;unction with debris derived from the host organism which is insoluble in the aqueous solution and wherein one or more extractions of denatured chymosin precursor are performed. In view of the relatively low cost of the materials involved, the alkali solubilisation technique is attractive in terms of commercial exploitation.
The methods of solubilisation in a strong denaturant such as guanidine hydrochloride or urea and solubilisation using alkali, each solubilise significant percentages of the insoluble chymosin precursor which are found in extracts from host organisms. However, neither is quantitative in terms of recovery o~ native chymosin precursor. The reasons for this have not been clearly defined and are probably different for the two types of ; solubilisation. It appears that guanidine hydrochloride solubilises all the material present but only a portion is converted into proteins capable of activation to chymosin.
Alkali treatment may not allow complete renaturation to form the native form of the chymosin precursor but in addition, does not solubilise all the insoluble form of the ~' , :

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chymosin precursor. We have discovered that by combining the two methods a greatly enhanced recovery of chymosin precursor in its native state may be obtained.
According to a further preferred aspect of the present invention the insoluble form of the chymosin precursor is denatured in an aqueous solution, the resulting solution is diluted into 10 to 50 volumes of an alkaline aqueous solution of between pH 9 and pH 11.5 and the chymosin precursor is renatured by reducing the pH of the solution below a pH effective to denature the chymosin precursor, to produce the soluble native form of the chymosin precursor.
j Preferably the solution containing the denatured chymosin precursor is diluted into an alkaline aqueous solution o~ pH between pH 10 and pH 11 and more pre~erably into an alkaline aqueoùs solution o~ pH from 10.5 to 10.9, pre~erably about 10.7.
The dilution introduces an element of physical separation between the denatured molecules, before renaturation is brought about, for example, by neutralisation of the alkaline denaturing solution. The dilution and resulting physical separation o~ the denatured molecules appears to assist their renaturation. The solubilisation process described immediately above leads to a recovery, in the case of methioniné-prochymosin, of more than 30% compared to, for example, 10 to 20~ for the multiple alkali extractions also described above.
Preferably, in the combined solubilisation process described above the insoluble form of the chymosin precursor is denatured in an aqueous solution comprising urea at a concentration o~ at least 7M or in a solution comprising guanidine hydrochloride at a concentration o~ at ~; least 6M.

`~ -~289~92 The present invention is preferably applied to the solubilisation of inso,1uble methionine-prochymosin produced by a host organism transformed with a vector including a gene coding for methionine-prochymosin.
Preferably the host organism is E. coli.
A process for preparing host organisms coding for a number of chymosin precursors, including methionine-prochymosin, is described in detail in our published British patent application GB~lO0737A. In addition published British patent application GB2100737A contains details for producing active chymosin from a chymosin precursor prepared in its native form by the solubilisation process of the present invention. Thus, for instance prochymosin can be activated to active chymosin in the presence of coli extract~ by exposure to acid pH. The extract is brought to p~2 at 20C by addition of lM hydrochloric acid and the mixture is agitated gently for l hour. ~ heavy precipitate forms which is removed by centrifugation at low speed. The clear Z0 supernatant is adjusted to pH6 by the addition of 2M
Tris pH 8 at 20C.
Some embodiments of the present invention are now described in detail by way of Examples.
Example l ,~ 25 An experiment was conducted in which the solubilisation of insoluble methionine-prochymosin produced by E. coli cells trans~ormed with vector pCT70 , was achieved using urea or guanidine hydrochloride as denaturant. The preparation of the transformed E. coli cell line is described in detail in published ,~
British patent application GB2lO0737A.
; Frozen ;E. coli/pCT70 cells grown under induced conditions were suspended in three tim~s their weight ~, of 0.05 M tris-HCl p~8, l mM EDTA, 0.233 M NaCl, 10%
glycerol (v/v) containing 130~g/ml of lysozyme and the suspension was incubated at 4 for 20 minutes.
' ,,~,~ Sodium deoxychloate was added to a final concentration ~.1 , , : . , ~: . . . . .
.

9~ -8a of 0.05~ and 10 ~g of DNAase 1 (from bovine pancreas) was added per gram of E. coli starting material. The solution was incubated at 15C for 30 minutes by which time the viscosity of the solution had decreased markedly.
An equal volume o a solution containing 0.01 M tris pH 8, 0.1 mM EDTA, 5~ glycerol was added and the extract centrifuged ~~

~2~ 2 for 45 minutes at 4C and 10000 x g. At this stage effectively all the methionine-prochymosin product was in the pellet fraction, presumably as a result of aggregation or binding to cellular debris. The pellet was washed in 50 volumes of 0.01 M tris-HCl, pH 8, o.l M NaCl, 1 mM EDTA at 4. After further centrifugation as above the supernatant solution was discarded and the pellet rapidly solubilised at room temperature in a buffer containing either 8 M urea or 6 M guanidine hydrochloride, 0.05 M tris. HCl pH8, 1 mM
EDTA and 0.1 M NaCl. It was then dialysed overnight at 4 against 200 volumes of 0.01 M tris. HCl pH8, 1 mM EDTA 0.1 M NaCl and 10% glycerol. A heavy precipitate formed (insoluble E.coll proteins~ which was removed by centrifugation to leave a solution of native methionine-pro¢hymosin protein. The soluble methionine-prochymosin ;; was in a form which could be converted catalytically to s active chymosin by acidification/
neutralisation, substantially as described in published British patent application GB2,100,737A.
Example 2 ~ An experiment was conducted in which the ; solubilisation of insoluble methionine-prochymosin produced by E.coli cells transformed with vector pCT70 was achieved using alkaline denaturation. The preparation of the trans~ormed E.coli cell line is described in detail in published British patent application GB2,100,~37A.
! Frozen E-coli/pCT70 cells grown under induced conditions were suspended in three times their own weight ..
of 0.05 M Tris-HCl pH 8, 1 mM EDrA, 0.1 M NaCl, containing 23 ~g/ml PMSF and 130 ~g/ml of lysozyme and the suspension was incubated at 4C for 20 minutes. Sodium deoxycholate was added to a final concentration o~ 0.5% and 10 ~g of DNA
ase 1 (from bovine pancreas) was added per gram of E.coli starting material. The solution was incubated at 15C for 30 minutes by which time the viscosity of the solution had decreased markedly. The extract, obtained as described ~' .~J

128~4~2 above, was centrifuged for 45 minutes at 4C and 10000 x g.
At this stage effectively all the methionine-prochymosin product was in the pellet fraction in insolubilised form, presumably as a result of aggregation or binding to cellular debris. The pellet was washed in 3 volumes of O.O1 M tris-HCl pH 8, 0.1 M NaCl, 1 mM EDTA at 4C. After further centrifugation, as above, the supernatant solution was discarded and the pellet resuspended in 3 volumes of alkali extraction buffer: 0.05 M K2 HP04, 1 mM EDTA, 0.1 M
NaCl, pH 10.7 and the suspension adjusted to pH 10.7 with sodium hydroxide. The suspension was allowed to stand for at least 1 hour (and up to 16 hours) at 4C, the pH of the suspernatant adjusted to 8.0 by addition of concentrated HCl and centrifuged as above. Methionine-prochymosin, representing a ~ubstantial proportion o~ the methionine-proch~mosin originally present in the pellet, was found to be present in the supernatant in a soluble form which could be converted to catalytically active chymosin by acidification/neutralisation activation treatment substantially as described in published British patent application GB2,100,737A.
We further noted that re-extraction of the debris left after the first alkali extraction liberates an equivalent amount of prochymosin. Alkali extraction may be repeate~ to a total of 4-5 times with the liberation of approximately equivalent levels of prochymosin at each extraction.
Example 3 An experiment was conducted in which the solubilisation of methionine-prochymosin produced by E.coli cells transformed with vector pCT70 was achieved using denaturation with guanidine hydrochloride, followed by dilution into an alkaline solution. The preparation of the transformed cell line is described in detail in published British patent application GB2,100,737~.

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1;~89~92 E.coli/pCT70 cell debris containing insoluble methionine-prochymosin was prepared and washed as described in Example 1 above and the following manipulations were carried out at room temperature. The cell debris was dissolved in 3-5 volumes of buffer to final concentration of 6M guanidine HCl/0.05 M Tris pH8, 1 mM EDTA, 0.lm NaCl and allowed to stand for 30 mins - 2 hrs. ThP mixture was diluted into 10-50 volumes of the above buffer at pH 10.7 lacking guanidine HCl. Dilution was effected by slow addition of the sample to the stirred diluent over a period of 10-30 minutes. The diluted mixture was readjusted to pH
10.7 by the addition of 1 M NaOH and allowed to stand for 10 mins - 2 hrs. The pH was then adjusted to 8 by the addition of lN HCl and the mixture allowed to stand for a further 30 minutes before centrifuging as above to remove precipitated proteins. The supernatant 80 produced contained soluble methionine-prochymosin which could be converted to catalytically active chymosin by acidification and neutralisation and purified as described in published British patent application GB2,100,737A. In a very similar experiment an 8M urea buffer was used in place of the 6M
guanidine HCl buffer described above. The results were as described above.

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Claims

1. A process for the production of chymosin in which an insoluble form of a chymosin precursor is produced by a host organism transformed with a vector including a gene coding for the chymosin precursor wherein the insoluble form of the chymosin precursor is solubilised to produce a soluble native form of the chymosin precursor prior to cleaving the soluble native form of the chymosin precursor to produce chymosin, the solubilisation involving reversibly denaturing the insoluble form of the chymosin precursor and subsequently allowing the chymosin precursor to renature, thereby forming the soluble native form of the chymosin precursor.

2. A process according to claim 1 wherein the insoluble form of the chymosin precursor is denatured in an aqueous solution comprising urea at a concentration of at least 7M and the chymosin precursor is renatured subsequently by reducing the concentration of urea in the solution below a concentration effective to denature the chymosin precursor to produce the soluble native form of the chymosin precursor.

3. A process according to claim 1 wherein the insoluble form of the chymosin precursor is denatured in an aqueous solution comprising guanidine hydrochloride at a concentration of at least 6M and the chymosin precursor is renatured subsequently by reducing the concentration of guanidine hydrochloride in the solution below a concentra-tion effective to denature the chymosin precursor, to produce a soluble native form of the chymosin precursor.

4. A process according to claim 1 wherein the insoluble form of the chymosin precursor is denatured in an alkaline aqueous solution of between pH 9 and pH 11.5 and the chymosin precursor is renatured subsequently by reducing the pH of the solution below a pH effective to denature the chymosin precursor, to produce the soluble native form of the chymosin precursor.

5. A process according to claim 4 wherein the insoluble form of the chymosin precursor is present in conjunction with debris derived from the host organism which is insoluble in the aqueous solution and wherein one or more extractions of denatured chymosin precursor are performed.

6. A process according to claim 1 wherein the insoluble form of the chymosin precursor is denatured in an aqueous solution, the resulting solution is diluted into 10-50 volumes of an alkaline aqueous solution of between pH 9 and pH 11.5 and the chymosin precursor is renatured by reducing the pH of the solution below a pH effective to denature the chymosin precursor, to produce the soluble native form of the chymosin precursor.

7. A process according to claim 6 wherein the insoluble form of the chymosin precursor is denatured in an aqueous solution comprising urea at a concentration of at least 7M.

8. A process according to claim 6 wherein the insoluble form of the chymosin precursor is denatured in an aqueous solution comprising guanidine hydrochloride at a concentration of at least 6M.

9. A process according to claims 1, 2 or 3, wherein the chymosin precursor is methionine-prochymosin.

10. A process according to claims 1, 2, or 3, wherein the host organism is E.coli.