Note: Descriptions are shown in the official language in which they were submitted.
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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
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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
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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
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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
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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
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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,
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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:
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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
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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
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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.
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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.
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TABLE 1
Effect of dilution on the recovery of milk clotting activity.
FOLD DILUTION MILK CLOTTING ACTIVITY
(mg)
5 UREA ALKALI OVERALL
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
