CA1212053A - Process for the production of a protein - Google Patents

Abstract

Abstract:
In a process for the production of a soluble native protein, such as immunoglobulin or methionine-prochymosin, in which an insoluble form of the protein is produced by a host organism transformed with a vector including a gene coding for the protein, the insoluble form of the protein is reversibly denatured in an alkaline aqueous solution at a pH selected to promote dissociation of a group or groups of the protein involved in maintaining the conform-ation of the protein, and the protein is subsequently allowed to renature by reducing the pH of the solution below a pH effective to denature the protein to produce the soluble native form of the protein. The pH of the alkaline aqueous solution is suitably in the range 9.0 to 11.5.

Description

~Zl~ 3 A process for the production of a protein Fieid of the Invention This invention relates to the field of protein production using recombinant DN~ biotechnology. In particular it relates to a process for the recovery of a protein produced in an insoluble form by a host organism transformed with a vector including a gene coding for the protein.

Background of the Invention There are now numerous examples of commercially valuable proteins which may be produced in large quantities by cultur-ing a host organism capable of expressing heterologous genetic material. Once a protein has been produced by a host organism it is usually necessary to treat the host organism in some way, in order to obtain the desired protein. In some cases, such as in the production of interferon in Escherichia coli a lysis or permeabilisation treatment alone may be sufficient to afford satisfactory yields. However, some proteins are produced within a host organism in the form of insoluble protein aggregates which are not susceptible to extraction by lysis or permeabilisation treatment alone. It has been ` ~Z~ 153 reported that a human insulin fusion protein produced in E.coli forms insoluble protein aggregates (see D.C. Williams et al (1982) Science 215 687-689).
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 three dimensional structure, the conformation of which is main-tained 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 the polypeptide chain, as well as inter-molecular disulphide bridges between separate polypeptide chains of multisubunit proteins, e.g. insulin. The insoluble proteins produced in some instances do not exhibit the func-tional activity of their natural counterparts and are there-fore 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 form. They may also possess un-wanted intermolecular disulphide bonds not required for func-tional activity of the native protein in addition to intra-molecular disulphide bonds. The altered three dimensional structure of such proteins not only leads to insolubility but also diminishes or abolishes the biological activity of the protein. It is not possible to predict whether a given pro-tein expressed by a given host organism will be soluble or insoluble.
In our published British Patent Application GB2100737A
we described a process for the production of the proteolytic enzyme chymosin. The process involves cleaving a chymosin precursor protein produced by a host organism which has been transformed with a vector including a gene coding for the relevant protein. In the course of our work we discovered that the chymosin precursor proteins were not produced in their native form but as an insoluble aggregate. In order :lZ~ S3 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 were solubilised and converted into their native form before the standard techniques of protein purii-cation and cleavage could be applied.
In our published International Patent Application WO 83/04418 the methods used for the solubilisation of chymosin precursor proteins are described. In general the techniques described involve the denaturation of the protein followed by the removal of the denaturant thereby allowing renaturation of the protein. In one example the dena~urant used is a com-pound such as urea or guanidine hydrochloride. When the in-soluble precursor is treated with urea or guanidine hydro-chloride it is solubilised. When the denaturant is removed, for example by dialysis, the protein returns to a thermo-dynamically stable conformation which, in the case of chymosin precursors, is a conformation capable of being converted to active chymosin.
The solubilised protein may be separated from insol-uble cellular debris by centrifugation or filtration. Theproduction of proteins from suitably transformed host organ-isms is potentially of great commercial value. The processes involved are of a type which may be scaled up from a labor-atory scale to an industrial scale. However, where the pro-tein produced is formed as an insoluble aggregate, potentialcomplications in the process may increase the cost of pro-duction beyond a viable level. The solubilisation technique described above, whilst effective to solubilise such proteins, is relatively expensive and may represent a significant pro-duction cost.
We have discovered a generally applicable solubili-sation process, which, in its broadest aspect, does away with the requirement of relatively expensive reagents.
- Summary of the Invention According to the present invention we provide a process for the production of a soluble native protein in which an ~Zi~S3 insoluble form of the protein is produced by a host organism transformed with a vector including a gene coding for the protein, wherein the insoluble form of the protein i8 revers-ibly denatured in an alkaline aqueous solution at a pH sel-ected to promote dissociation of a group or groups of theprotein involved in maintaining the conformation of the pro-tein and the protein is subsequently allowed to renature by reducing the pH of the solution below a pH effective to de-nature the protein to produce the soluble native form of the protein.
The use of an alkali solution to denature the insoluble protein reduces the reagent cost of the process. The pH is selected with reference to the protein to which the process is to be applied. In particular the pH is selected such that groups responsible for holding the protein in an un-natural conformation by means of intramolecular, or poten-tially in the case of a protein aggregate nonfunctional inter-molecular, bonds or forces are dissociated such that, when the pH is reduced, the protein refolds in the native con-formation. The groups responsible for holding the protein inan unnatural conformation may be ionisable groups, in which case the pH is preferably selected to be compatible with the pXa of the relevant ionizable group.
Studies in our laboratory have shown that intermolecular disulphide bonds exist in prochymosin aggregates produced in E.coli (Schoemaker et al ~19~4) submitted to PNAS). Native prochymosin is monomeric and contains three intramolecular disulphide bonds (Foltmann et al (1977) Proc. Natl. Acad.
Sci. U.S.A. 74 pp 2321-2324). Six thiol groups per molecule are therefore available to form intermolecular and intra-molecular bonds within the protein aggregate. Consequently, disulphide bonds must be broken and correctly reformed for denaturation/renaturation to successfully solubilise pro-chymosin. This may be achieved by using an alkaline aqueous solution of pH10.7 (+0.5). The free thiol groups ox cysteine have a pXa value of 10.46.
The term "insoluble" as used herein means in a form which, under substantially neutral conditions (for example I, - ~L2~L~f~S3 pH in the range 5.5 to 8.5), is substantially insoluble or is in an insolubilised association with insoluble material pro-duced 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 aggre-gates or may simply be associated with insoluble cell membrane material. The process permits the separation of solubilised protein from insoluble cellular debris.
Any suitable alkali may be used in the process, for example an aqueous solution of an alkali metal hydroxide such as NaOH or KOH, an aqueous buffer, or an aqueous solution of an organic base such as triethylamine.
Preferably the alkaline aqueous solution has a pH of from 9 to 11.5, most preferably from 10 to 11.
The treatment of an insoluble protein with an alkaline aqueous solution may not, in all cases, result in complete solubilisation of the protein. Since insoluble material is present at all times, a number of mass transfer effects may be 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 advantage of minimising the time for which the solubilised protein is in contact with alkali. Preferably, therefore, one or more extractions of denatured protein are performed.
The methods of solubilisation in a strong denaturant such as guanidine hydrochloride or urea described in pub-lished British patent application GB2100~37A and in published International patent application WO 83/04418 and the methods of solubilisation using alkali, according to the broad aspect of the present invention each solubilise significant per-centages of insoluble proteins found in extracts from host organisms. However, neither is completely quantitative in terms of recovery of native protein. 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 native proteins after removal of ;3 guanidine hydrochloride. A:Lkali treatment may not allo~7 com-plete renaturation to form the native form of the protein and in addition does not solubilise all of the insoluble form of the protein. We have discovered that by combining the two methods a greatly enhanced yield of native protein may be ob-tained.
According to a preferred aspect of the invention the insoluble form of the protein is first denatured in an aqueous solution, and subsequently the resulting solution is diluted in an alkaline aqueous solution at a pH selected to promote dissociation of the group or groups of the protein involved in maintaining the conformation of the protein and the protein is renatured by reducing the pH of the solution below a pH
effective to denature the protein, to produce the soluble native form of the protein.
The dilution introduces an element of physical separa-tion between the denatured molecules, before renaturation is brought about for example, by neutralisation of the alkaline denaturing solution. The dilution and resulting physical separation of the denatured molecules appears to assist their renaturation in native form. The solubilisation process described immediately above leads to a recovery, in the case of-methionine-prochymosin, of more than 30% compared to, for example, 10 to 20% for the multiple alkali extractions also described above.
Preferably the pH of the alkaline aqueous solution is from 9 to 11.5, most preferably from 10 to 11.
Preferably the dilution is from 10 fold to 50 fold.
tThat is a dilution into a total volume of from 10 to 50 volumes).
Preferably, in the combined solubilisation process described above the insoluble protein is denatured in an aqueous solution comprising urea at a concentration of at least 7M or in a solution comprising guanidine hydrocloride at a concentration of at least 6M.
The insoluble protein may be a recombinant animal protein produced by a host organism. Examples of such pro-teins are immunoglobulin light and heavy chain polypeptides, foot and mouth disease antigens and thymosin and insulin pro-teins.
The host organism may be a naturally occuring organism or a mutated organism capable of producing an insoluble pro-tein. Preferably, however, the host organism is an organismor the progeny of an organism which has been transformed using recombinant DNA techniques with a heterologous DNA sequence which codes for the production of a protein heterologous to the host organism and which is produced in an insoluble form.
The host organism may be a eukaryotic organism such as a yeast or animal or plant cell. Preferred yeasts include Saccharomyces cerevisiae and kluyveromyces. In the alter-native the host organism may be a bacterium such as E.coli, B. subtilis, B. stearothermophilis or Pseudomonas. Examples of specific host organism strains include E.coli HB101, E.coli X1776, E.coli X2882, E.coli PS410, E.coli RV308 and E.coli MRCl.
- The host organism may be transformed with any suitable vector molecule including plasmids such as colEl, pC~l, pBR322, RP4 and phage DNA or derivatives of any of these.
Prior to treatment with the process of the present in-vention the host cells may be subjected to an appropriate lysis or permeabilisation treatment to facilitate recovery of the product. For example, the host organism may be treated with an enzyme, for example a lysozome, or a mechanical cell destructing device to break down the cells.
The process of the invention may then be employed to solubulise the insolubilised product and the resulting solu-tion may be separated from solid cell material such as in-soluble cell membrane debris. Any suitable method includingfiltration or centrifugation may be used to separate solution containing the solubilised protein from the solid cell mate-rial.
The present invention is now illustrated by way of the following Examples:

I, i3 Example 1 An experiment was conducted in which the solubili-sation of insoluble methionine-prochymosin produced by E.coli cells transformed with vector pCT70 was achieved using alkaline denaturation. The preparation of the transformed E.coli cell line is described in detail in published British patent application GB2100737A.
Frozen E.coli/pCT 70 cells grown under induced con-ditions were suspended in three times their own weight of 0.05 M Tris-HCl pH 8, 1 mM EDTA, 0.1 M NaCl, containing 23 ~g/ml phenylmethylsulphonylfluoride (PMSF) and 130 ~g/ml of lysozyme and the suspension was incubated at 4C for 20 minutes. Sodium deoxycholate was added to a final con-centration of 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 above, was centrifuged for 45 minutes at 4C and 10000 x g. At this stage effect-ively 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 0.01 M tris-HCl, pH8, 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 HPO4, 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 Rand up to 16 hours at 4C, the pH of the supernatant adjusted to 8.0 by addition of concentrated HCl and centrifuged as above. Methionine-prochymosin, representing a substantial proportion of the methionine-prochymosin 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 lJS3 g as described in published British patent application GB2100737A.
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 repeated to a total of 4-5 times with the liberation of approximately equivalent levels of prochymosin at each extraction.

Example 2 An experiment was conducted in which the solubilisation of an insoluble immunoglobulin light chain polypeptide produced by E.coli cells transformed with vector pNP3 was achieved using alkaline denaturation. The preparation of the transformed E.coli cell line is described in International Patent public-ation No. WO 84/03712, puplished September 27, 1984. E.coli cells transformed with the plasmid pNP3, containing a gene coding for the Al light chain of the 4-hydroxy-3-nitrophenyl acetyl (NP~ binding monoclonal antibody S43, were grown under inducing conditions. The cells were harvested and resuspended in 0.05M TRIS pH 8.0, 0.233 M NaCl. 5% glycerol v/v containing 130 ~g/ml of lysozyme and incubated at 4C or room temperature for 20 minutes. Sodium deoxycholate was then added to a final concentration of 0.05% andlO ~g of DNA ase 1 (from bovine pan-creas) was added per gm wet wt of E.coli. The solution was then incubated at 15C for 30 minutes by which time the viscos-ity of the solution had decreased markedly. The resultant 25 mixture was then centrifuged (at 10,000 x g for 15 minutes for small volumes l ml) or 1 ho~lr for larger volumes). Im-munoprecipitation studies indicated that the A light chain protein was present in the insoluble fraction rather than the soluble fraction.
In order to purify the recombinant light chain, the E.coli pellet fraction, obtained as described above, was re-suspended in a pH 11.5 buffer comprising 50 my K2 HPO~, 0.1 M NaCl and 1 mM EDTA. The suspension was allowed to stand for at least 1 hour (and up to 16 hours), centrifuged as above and the pH of the supernatant adjusted to 8.0 by ,Zl~i3 _ 10 --addition of concentrated HC1. A substantial proportion of the light chain protein, originally present in the pellet was found to be present in the supernatant is a soluble form.

Example 3 An experiment was conducted in which the solubilï-sation of methionine-prochymosin produced by E.coli cells transformed with vector pCT70 was achieved using:denatura-tion with guanidine hydrochloride, followed by dilution into an alkaline solution. The preparation of the trans-formed cell line is described in detail in published British patent application GB2100737A.
E.coli/pCT 70 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 dis-solved in 3-5 volumes of buffer to final concentration of 6M
guanidine HC1~0.05 M Tris pH8, 1 mM EDTA, 0.1 m NaCl and allowed to stand for 30 minutes - 2 hours. The mixture was diluted into 10-50 volumes of the above buffer at pH 10.7 lacking guanidine HCl. Dilution was effected by slow addi-tion of the sample to the stirred diluent over a period of 10-30 minutes. The diluted mixture was readjus$ed to pH 10.7 by the addition of 1 M NaOH and allowed to stand for 10 minutes - 2 hours. 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 so produced contained soluble methionine-prochymosin which could be converted to catalytically active chymosin by acidification and neutral-isation and purified as described in published British patentapplication GB2100737A. 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.

.~

~Z~ 3 Example 4 An experiment was conducted in which the solubilisation of methionine-prochymosin produced by E.c~li cells trans-formed with vector pCT 70 was achieved using a method similar to that described in Example 3. The preparation ox the trans-formed cell line is described in detail in published British patent application GB2100737A.
E.coli/pCT 70 cell debris containing insoluble methionine-prochymosin was prepared and washed as described in Example 1 above and the following manipulations were car-ried out at room temperature. The washed pellets were sus-pended in a buffer containing 50 mM Tris HCl pH 8.0, 1 mM EDTA, 50 mM NaCl and 0.1 mM PMSF supplemented with 8 M urea (deionized). For every 10 g weight of starting material, 90 ml of buffer were used.- After 1 hour, this solution was added slowly to a buffer containing 50 mM KH2PO4 pH 10.7 containing 1 mM EDTA and 50 mM NaCl and left for at least 30 minutes.
Various dilutions were made in order to establish an optimum dilution range. This proved to be a dilution in alkali of between 10 and 50 fold. The pH was maintained at pH ]0.7 for the period and then adjusted to pH 8. The product was acti-vated to give active chymosin and the level of chymosin activ-ity was assayed (Emtage, J.S., Angal S., Doel, M.T., Harris, T.J.R., Jenkins, B., Lilley, G., and Lowe, P.A. (1983) Proc.
Natl. Acad. Sci. USA 80, 3~71-3675). The results are shown in Table 1.

;i3 Effect of dilution on the recovery of milk clotting activity.

FOLD DILUTION MILK CLOTTING ACTIVITY
(mg) 1 10 10 0.04 1 25 25 0.10 l 50 50 0.05 0.10 10 5 25 125 0.09 250 1.23 100 1.97 250 1.56 500 0.09 Example 5 An experiment was conducted in which the solubilisation of insoluble immunoglobulin heavy and light chains produced together in E.coli was achieved using denaturation with urea followed by dilution into alkali. The preparation of the transformed cell line is described in International patent publication No. WO 84/03712.
In order to produce functional antibodies from E.coli cells expressing the genes for both the heavy and light immunoglobulin chains, the cells were lysed, the insoluble -material was washed followed by sonication (three times for 3 minutes). The material was then dissolved in 9 M urea Glycine-Na~ pH10.8 1 Mm EDTA and 20 mM 2-mercaptoethanol.
This extract was dialysed for 40 hours against three changes of 20 vols. of 100 mM ECl 50 Mm Glycine-Na+ pH10.8 5~ glycerol 0.5 mM EDTA 0.5 mM reduced glutathione and 0.1 mM oxidised glutathione. The dialysate was cleared by centrifugation at 30,000 g for 15 minutes and loaded directly onto DEAE
sephacel followed by development with 0-0 5 M KCl linear gradient in 10 mM Tris-HCl 0.5 mM EDTA pH8Ø

Claims (8)

CLAIMS:

1. A process for the production of a soluble native protein in which an insoluble form of the protein is produced by a host organism transformed with a vector including a gene coding for the protein, wherein the insoluble form of the protein is reversibly denatured in an alkaline aqueous solution at a pH
selected to promote dissociation of a group or groups of the protein involved in maintaining the conformation of the pro-tein, and the protein is subsequently allowed to renature by reducing the pH of the solution below a pH effective to de-nature the protein to produce the soluble native form of the protein.

2. A process according to claim 1 wherein the alkaline aqueous solution has a pH from 9 to 11 5.

3. A process according to claim 1 or 2 wherein the in-soluble form of the protein is present in conjuction with debris derived from the host organism which is insoluble in alkaline aqueous solution and wherein one or more extrac-tions of denatured protein are performed.

4. A process according to claim 1 wherein the insoluble form of the protein is first denatured in an aqueous solution, and subsequently the resulting solution is diluted into an alkaline aqueous solution at a pH selected to promote dis-sociation of a group or groups of the protein involved in maintaining the conformation of the protein and the protein is renatured by reducing the pH of the solution below a pH
effective to renature the protein, to produce the soluble native form of the protein.

5. A process according to claim 4 wherein the pH of the alkaline aqueous solution is from 9 to 11.5.

6. A process according to claim 4 wherein the dilution is from 10 fold to 50 fold.

7. A process according to claim 4, 5 or 6 wherein the in-soluble protein is denatured in an aqueous solution compris-ing urea at a concentration of at least 7 M.

8. A process according to claim 4, 5 or 6 wherein the in-soluble protein is denatured in an aqueous solution compris-ing guanidine hydrochloride at a concentration of at least 6 M.