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Description  |
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FIELD OF THE INVENTION
This invention relates to an improved process for the production of
polypeptides produced by genetic engineering techniques.
BACKGROUND OF THE INVENTION
Recombinant DNA technology has now introduced the possibility of producing,
for example, pharmacologically-active proteins and peptides in
microorganisms. Furthermore, it is possible to introduce changes by means
of gene cloning such that the resulting polypeptides or proteins have
improved or modified biological activity or stability as compared to the
native gene product. However, prior to biological testing and clinical
use, it is essential that the peptides or proteins should be purified to a
very high degree in order to remove contaminating bacterial proteins,
nucleic acids and endotoxins which may cause deleterious side effects.
Therefore, there is a need for enhanced methods of purifying proteins
produced using recombinant DNA techniques.
Currently, there are numerous methods available to purify peptides and
proteins, e.g. affinity, ion-exchange, hydrophobic and molecular sieve
chromatography, (see, for example, Williams, B. L., and Wilson, K.,
Principles and Techniques of Practical Biochemistry, (1975), Edward
Arnold, London, 28-123). In order to achieve pure product in high yields
and at reasonable cost, considerable development of such methods is
necessary. Furthermore, at the moment, different methods must be developed
and optimised for each new product. Even small changes in amino acid
composition may alter the purification properties such that a modified
purification procedure will need to be developed.
A further difficulty in the development of new products by recombinant DNA
technology is the assay of the product. Many of the proteins and peptides
have no enzymatic activity and may only be determined by either the in
vitro or in vivo biological activity thereof. Such assays tend to be
inaccurate and time consuming, while purification strategies require large
numbers of highly accurate assay results. Immuno-assays based on the
highly specific recognition of a protein by an antibody may provide such
accurate and rapid assays, (see, for example, Eisen, H. N., Immunology,
(1974), Harper and Row, U.S.A., 395-396). However, the raising of antisera
to a protein is best achieved by inoculating animals with purified antigen
and considerable expertise and time needs to be spent on this task. Also,
not only would new antisera need to be raised for each new recombinant
product, but because of the high specificity of these antibodies, even
small modifications in the amino acid sequence may alter the binding of
the product to the antibody and reduce the accuracy of the results.
One approach has been to fuse cloned peptides with a native bacterial
protein, e.g. .beta.-galactosidase (.beta.-gal) and .beta.-lactamase (see,
for example, Davis A. R., et al, Proc. Natl. Acad. Sci. U.S.A., (1981),
78, No. 9, 5376-5380; and published European Patent Application No.
35384). Hybrids may then acquire all of the properties of native protein,
e.g. convenient assay, established purifications and, in the latter case,
secretion from the host cell. However, in the case of
.beta.-galactosidase, (.beta.-gal), which is a high molecular weight
tetramer, the correct association of the .beta.-gal hybrid subunits may be
altered or prevented by the tertiary structure of the hybrid. Although
this does not occur when low molecular weight peptides are fused to
.beta.-gal, there is no reason to assume that larger and structurally more
complex hybrid proteins will still allow the correct association of
subunits to form a fully active enzyme. Without subunit association,
recombinants contained the fused polypeptide would not be identified by
the .beta.-gal assay. In a similar manner, the alteration in secondary or
tertiary structure of a .beta.-lactamase fused protein may prevent
secretion thereof.
For clinical use, the cloned peptide or protein must be cleaved from the
hybrid. Chemical cleavage at methionine residues has been described, but
this is of limited use for most peptides and proteins, (see, for example,
Goeddel, D. V. et al, Proc. Natl. Acad. Sci. U.S.A., (1979), 76, No. 1,
106-110). To this end, it has been suggested that, by introducing the
correct peptide sequence, an endopeptidase might be used to specifically
cleave the .beta.-gal protein from the desired peptide or protein, (see,
for example, published European Patent Application No. 35384). For this
approach to work, not only must this cleavage site be unique in the cloned
protein or peptide, but also the folding of the entire fused protein must
be such that the cleavage site is available to the endopeptidase. Such
fused proteins would also share few similarities with the native
endopeptidase substrate and the rate of cleavage may be considerably
reduced. Furthermore, such endopeptidases could leave amino acids from the
cleavage site on the protein of interest thereby making the protein
unsuitable for many purposes.
Published European Patent Application No. 35384 related to DNA sequences
coding for amino acid sequences which contain specific cleavage sites.
These DNA sequences could be attached to a cloned DNA coding sequence.
According to this reference, particularly the amino terminus of an
expressed protein may be provided with a removable terminal sequence
having distinctive physical properties which are useful for purification.
Here it was important that the junction be provided with a cleavage site
for an endopeptidase. In an attempt to approach the desired specificity,
this prior art advocated the use of extended recognition sites for unusual
enzymes. Of course, this procedure would have to be adapted to each
protein and subject to the above limitations.
On the contrary, the present improved approach does not depend in the same
way on the structure of the product. By virture of the use of an
exopeptidase, the problem of simultaneous cleavage of the product is
obviated without the need for complicated recognition sites for unusual
enzymes. More importantly, unlike an endopeptidase, an exopeptidase will
not hydrolyze the polypeptide product internally. The present system
enjoys a further advantage in that the possibility of assay of the product
is provided, which is not foreshadowed in the prior art. In the present
case, attention is particularly directed to the carboxy terminus.
The present invention discloses a surprisingly useful process requiring a
charged amino acid polymer and an exopeptidase that selectively removes
the polymer and that does not harm the desired polypeptide product. The
present invention includes any terminal amino acid polymer and the present
invention also incudes the use of any exopeptidase, including both
aminopeptidases and carboxypeptidases. The polymer may be at either (or
both) the amino or carboxy terminals of the desired polypeptide product.
Unlike the prior art the present invention allows via genetic engineering
the attachment of an easily isolated polypeptide to a protein of interest
followed by the selective removal of the attached polypeptide without harm
to the protein of interest. In addition, the attached polypeptide serves
as an easily quantitated tag reducing the requirement for expensive and
difficult bioassays of the protein or its activity. A structural gene is
defined as any gene coding for a polypeptide.
SUMMARY OF THE INVENTION
An object of the present invention is to improve the yield and purity of
polypeptide products by the synthesis of peptides containing amino acid
polymer ends which can be selectively removed by the action of
exopeptidases.
An object of the present invention is to utilize recombinant DNA technology
and gene synthesis so as specifically to modify cloned proteins to result
in improved purification, stabilization and assay. In general terms, this
may be achieved by the introduction of tailored peptides of known sequence
at either or both end(s) of the cloned protein.
An object of the present invention is to provide a method of producing a
specific polypeptide involving a structural gene contained in a cloning
vehicle such as a plasmid expressed in a prokaryotic cell and the
isolation of the said structural gene product comprising:
(a) inserting adjacent to the said structural gene at either end a DNA
sequence coding for a polymer of charged amino acids;
(b) expressing the said structural gene product and charged amino acid
polymer as a fusion product;
(c) separating the said fusion product from contaminants based upon the
properties of the said polymer of charged amino acids;
(d) removing the said polymer of charged amino acids using an exopeptidase;
and optionally, if further purification is required,
(e) isolating the said structural gene product free of the said polymer of
charged amino acids.
As shown in the examples polyarginine is one charged amino acid polymer or
"tag" useful in the present invention.
In addition to the polyarginine "tag" other amino acids may be used as a
peptide "tag". Peptide "tags" may be designed to include amino acids which
modify, for example, the hydrophobicity, the charge or the chemical
properties of the product. This allows the production of a peptide
conjugate having unusually high affinity on, respectively, hydrophobic
interaction, ion-exchange or covalent chromatography, for example.
Furthermore, certain characteristics of the peptide tag may be used for
affinity chromatography or gel filtration or, for example, dye ligand or
antibody columns, (see, for example, Lowe, C. R., and Dean, P. D. G.,
Affinity Chromatography, (1974), John Wiley and Sons, New York).
Thus, the addition of the peptide tag allows the prediction of specific
purification properties of the fused polypeptide and so the rapid
purification of the cloned product may be achieved.
Also, the present invention encompasses the use of further "negative"
purification techniques. The specific removal of the peptide tag means
that the contaminating products that were co-purified with the desired
conjugate using, for example, chromatography step, will still be made to
bind to the same chromatography column, while the cloned peptide or
protein will not.
The added peptide sequence may be required to be removed to assist in the
purification and to provide material for biological testing and clinical
use. This may be achieved by including specific amino acids or amino acid
sequences within the tag which are susceptible to the enzymatic cleavage.
In general, enzymatic hydrolysis may be achieved using either an
endopeptidase or an exopeptidase or combinations of these enzymes.
However, in accordance with the present invention, greater specificity and
control of hydrolysis is achieved using an exopeptidase, either an
aminopeptidase or a carboxypeptidase. By the use of enzymes with defined
specificity it is possible to limit the amount of peptide removed from
either the carboxy terminus or the amino terminus until the required
protein or peptide is produced, (see, for example, Ambler, R. R., Methods
in Enzymology, (1968), Academic Press, New York, 11, 155-166). For
example, carboxypeptidase B is specific for the basic amino acids arginine
and lysine and will not digest other carboxy-terminal amino acids at a
significant rate. In contrast, carboxypeptidase A has a wider substrate
specificity but it digests arginine and lysine very poorly. Hence, for
example, by the complimentary action of these two readily available and
well characterized carboxypeptidases, very careful control over the
digestion of a carboxy-terminus can be achieved. In doing this the
carobxypeptidases can be used either simultaneously or sequentially to
digest the carboxy-terminus. Furthermore, as it is highly unlikely that
any contaminating proteins will have the particular sequence in the
peptide tag at the amino or carboxy ends, very little alteration in their
properties, therefore will occur. Therefore, the negative purification
step described above may be used to greater effect.
The efficiency of the exopeptidase may be increased in large scale
processes by recycling using enzyme reactor systems. The reaction may be
monitored by simple assay techniques, e.g. potentiometrically, (see, for
example, Walsh, K. A., and Wilcox, P. E., Methods in Enzymology, (1970),
Academic Press, New York 19, 35-38), and the efficiency checked by
identification of released amino acids, or peptides using, for example, an
amino acid analyser, (see, for example, Light A., Protein Structure and
Function, (1974), Prentice Hall Inc., New Jersey, 107-121).
It is also advantageous that the peptide tag should not prevent a
recombinant protein from assuming its native conformation. By fusing the
peptide tag at the carboxy-terminus, the polypeptide folding is less
influenced by the additional amino acid sequence, (see for example,
Wetlaufer, D. B., Advances in Protein Chemistry, (1981), Academic Press,
London and New York, 61-92). A carboxy-terminal protein tag also has the
advantage that only those proteins or peptides which have the complete
peptide sequence will acquire both the predicted purification properties
and assay characteristics.
It is preferable that the peptide tag should be readily accessible to a
purification matrix in an aqueous environment, that is, on the surface of
the protein. Therefore, a hydrophilic structure would be preferred. It is
also preferred that the tag should not undergo chemical reactions with
other parts of the molecule, e.g. an excess of thiol groups might affect
the cross-linking of a protein or cause polymerisation of the conjugate.
Therefore, it is preferred to alter the charge properties by using, for
example glutamic acid or lysine, rather than the hydrophobic (e.g. using
leucine) or chemical (e.g. using cysteine) properties of the conjugate.
The majority of proteins from bacteria are acidic or negatively charged as
are nucleic acids and pyrogens. It is therefore, preferred for the peptide
tag to confer a net positive charge on the conjugate so that these
contaminants may be rapidly removed by ion-exchange chromatography. Of the
three positively-charge amino acids, histidine, lysine and arginine, the
last-mentioned is the most hydrophilic and highly charged, (see, for
example, Wolfenden, R., et al, Biochemistry, (1981), 20, 849-855). It is,
therefore, particularly advantageous for the peptide tag to be rich in
arginine residues.
The composition of the tag may be varied as desired to suit particular
requirements. However, as regards purification and ease of removal, it is
preferable to have a simple polymeric structure, e.g. arginine-lysine, or
more preferably, a homopolymer of arginine alone, e.g. poly-arginine.
The length of the peptide tag should be such that it does not result in an
excessive amount of the cultures nutrients and amino acid pools being used
for its biosynthesis or in translation problems or in plasmid instability.
It must be long enough, however, to confer the desired properties on the
protein. In the case of a homopolymer, some restriction on length will be
caused by the availibility of tRNA for the homopolyme fragment which may
adversely affect protein translation. Again, arginine is particularly
useful because six possible codons are available. These DNA triplet codons
coding for arginine are CGT, CGC, CGA, CGG, AGA and AGG. They can be used
to construct a DNA polymer coding for the synthesis of polyarginine. The
DNA sequence coding for a polymer of charged amino acids may be 2 to 30
consecutive triplet codons, 3 to 12 consecutive triplet condons or 5
consecutive triplet codons. For arginine polymers this would require 2 to
30, 3 to 12, or 5 consecutive arginine triplet codons. This DNA may
contain either mixed triplet codons with the advantage of reduced tRNA
limitations or one triplet codon repeated, depending upon the optimum
requirements for utilizing the cell's translation system most efficiently.
Bearing in mind these constraints, a peptide tag length of from 2 to 30
amino acids, preferably from 3 to 12 amino acids, most preferably 5 amino
acids may be used.
Assay of the product may be achieved by specific chemical characteristics
of the tag. These include chemical reactivity (e.g. arginine-rich peptides
may be assayed using the Sakaguchi reagent (Sakaguchi, S, J. Bio chem 5,
33 1925), or cysteine-rich peptides by sulphhydrol group reactivity), UV
adsorption (e.g. in the case of tryptophan) or specific dye adsorption
properties (e.g. for poly-arginine, see, for example, Itzhaki, R. F.,
Anal, Biochem. (1972), 50, 569-574.)
In addition, antibodies, including monoclonal antibodies, may be raised to
the peptide tag and used in competitive binding assays. Again, a
homopolymer is the simplest antigen. These may be raised by coupling the
peptide tag to an antigenic protein. Specific antibodies to the peptide
tag may then be selected, (see, for example, Geller, S., et al,
Biochemistry, (1980), 19, 3614-3623). The fused protein may then be
assayed using immuno-assays, e.g. RIA (radio-immuno-assay) or ELISA
(enzyme-linked-immunosorbant-assay). This assay would then be suitable for
any other products containing the "tag" which would minimize assay
development efforts.
By incorporating the correct sequence of amino acids, it is also possible
to protect sensitive proteins from degradation. For example, small
hydrophobic peptides may be protected from surface adsorption or
denaturation by the presence of a protective charged tag. Protease
sensitivity caused by specific or nonspecific bacterial proteases may be
reduced by the inclusion of an inhibitory sequence of amino acids. For
example see FIG. 1 incorporating polyarginine. These protective sequences
may be incorporated into the peptide tag or fused to the other end of the
cloned protein from that fused to the peptide tag and removed as described
above.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 expression of the urogastrone gene in E. coli with and without a
polyarginine tail.
FIG. 2 the assay of polyarginine-tailed urogastrone by the action of
carboxypeptidase B releasing primary amines.
FIG. 3 the elution pattern of urogastrone with and without a polyarginine
tail on SP-Sephadex.RTM..
DETAILED DESCRIPTION OF THE INVENTION
A polymer containing highly charged amino acids, when covalently attached
to a polypeptide of interest, serves as a very useful "tag" for the
stabilization, isolation and assay of the polypeptide. By attaching a DNA
sequence coding for such an amino acid polymer to the gene coding for the
polypeptide of real interest, the polypeptide product is synthesized with
the amino acid "tag" attached. This "tag" may be either positively or
negatively charged at physiological pH. This "tag" may be at either the
amino or carboxy terminus of the polypeptide of interest.
The presence of this amino acid "tag" results in stabilization of the
polypeptide product, particularly against the action of proteases.
By selecting on the basis of charge, the polypeptide-amino acid tag fusion
product can be easily separated from contaminants. Ion exchange
chromatography, electrophoresis and isoelectric focusing are among the
methods that can be used to isolate the fusion product.
Larger amino acid tags may be needed when larger polypeptide products are
to be produced. Similarly, when the polypeptide product has only a slight
charge or is neutral a larger charged amino acid polymer may be required.
Removal of the charged amino acid polymer is effected using exopeptidases
rather than endopeptidases. Exopeptidases hydrolyze the terminal amino
acids of the fusion product sequentially from either the amino or carboxy
terminus. Often, the exopeptidases are specific for charged amino acids,
resulting in an end to the terminal hydrolysis of the fusion product as
soon as the charged amino acid polymer is removed from the fusion product.
This results in the release of the polypeptide unharmed and available. One
example of such an exopeptidase is carboxypeptidase B which catalyzes the
hydrolysis of the basic amino acids lysine and arginine from the carboxyl
terminal position in polypeptides.
As an illustration of how such a charged polymer of amino acids is useful
in the production of a polypeptide the following experimental system is
presented with urogastrone as the polypeptide, polyarginine as the charged
amino acid polymer "tag" and carboxypeptidase B as the exopeptidase. Other
polypeptides, amino acid tags and exopeptidases can also be used based
upon the preceding discussion. They will also function in an analogous
manner for polypeptide production.
By conventional nucleotide synthesis techniques, a DNA segment was
synthesized which corresponds to the C-terminal portion of urogastrone
with additional arginine residues following the native C-terminus, in this
case five arginines, which is equivalent to .gamma.-urogastrone (which has
similar properties to .beta.-urogastrone except the carboxy terminal
arginine is missing) with an additional six arginines. This DNA segment
incorporated two restriction sites, one toward the 5' and one at the 3'
ends thereof, as well as a stop signal following the fifth arginine. This
piece of synthetic DNA was cloned into E. coli containing a recombinant
urogastrone gene thereby altering the resulting urogastrone C-terminus to
contain a polyarginine tag.
The expressed urogastrone protein with its arginine tag was purified and
assayed by selectively utilizing the properties of the polyarginine tag.
Purification was effected by means of ion-exchange chromatography on
CM-Sepharose CL-6B or SP-Sephadex.TM.. Following initial purification,
virtual homogeneity was achieved by specific removal of the poly-arginine
tag and rechromatography on SP-Sephadex. (See Table 1 and 2 and FIG. 3)
Efficient removal of the tag was achieved by incubating with immobilized
carboxy peptidase B (CPB). (See FIG. 2 and Table 3)
The specificity of the CPB reaction was utilized to assay the expressed
protein by determination of arginine released upon hydrolysis with CPB.
The addition of a five amino acid polyarginine tail at the carboxy terminus
of .beta.-urogastrone resulted in more than a 3-fold increase in
detectable urogastrone synthesized in E. coli. This increase in
urogastrone might be due, in part, to a decreased rate of protease
activity in the presence of the polyarginine.
This additional synthetic DNA sequence was inserted into the
.beta.-urogastrone gene which resulted in an additional five arginines at
the carboxy-terminus of urogastrone. This was achieved by inserting at the
28 base pair terminal Bgl II-Bam H1 sequence of the synthetic urogastrone
gene, a 43 base pair sequence coding for a new carboxy terminus which
resulted in a urogastrone protein with the required polyarginine tail. The
DNA nucleotide sequence inserted and its corresponding amino acid sequence
and restriction sites are shown below.
______________________________________
asp leu lys trp trp glu
5' GAT CTG AAA TGG TGG GAG
(restriction site)
3' AC TTT ACC ACC CTC
leu arg arg arg arg arg
CTC CGT CGA CGC CGT CGA
GAG GCA GCT GCG GCA GCT
arg stop
CGT TAA G 3'
GCA ATT CCT AG 5' (Restriction site)
______________________________________
To produce quantities of urogastrone this gene coding for urogastrone has
been constructed and cloned into E. coli using conventional recombinant
DNA techniques. Expression of the urogastrone gene was achieved using an
expression plasmid utilizing the E. coli tryptophan promotor producing a
hybrid polypeptide with an additional 14 amino acids at the
amino-terminus. The methods used for the expression plasmid were described
by Tacon et al. (1980) Molec. Gen. Genet. 177, 427-438 and the methods for
the hybrid polypeptide were described by Smith et al. (1982) Nucleic Acids
Res. 10, 4467-4482, both articles herein incorporated by reference.
The polyarginine tail also changes the migration of the
urogastrone-polyarginine hybrid during electrophoresis. This property
allows selective purification of the urogastrone analogous to that
achieved using ion-exchange chromatography. Similarly, polyarginine
specific antibody, either polyclonal or monoclonal, can be used to
specifically bind polyarginine-tailed peptides. Through the use of a three
step purification process, using either electrophoresis, affinity
chromatography or antibody binding as the selective procedure, a pure
urogastrone can be achieved. That is, a first step separating polyarginine
containing polypeptides of interest followed by a second step where the
polyarginine is selectively removed and a third step where the separation
process is repeated if necessary. The second separation step removes
contaminating molecules that do not have an exopeptidase-sensitive
polyarginine region, but which do exhibit polyarginine-like properties
during the selective procedure. The result of the three step procedure,
separation, exopeptidase and separation, results in essentially pure
polypeptide of interest free of the polyargine tail.
Proteins other than urogastrone can be prepared using the isolation
procedures described. These other proteins incude peptide hormones,
enzymes, blood clotting factors, antibodies or antibody fragments, peptide
cofactors, cytochromes, plasma proteins, structural proteins, peptide
antigens, antibiotics and other polypeptides of interest. Among the
peptide hormones of interest are the interferons, follicle stimulating
hormone (FSH), leukotreins, luteninizing hormone (LH), prolactin, growth
hormone, thyroid stimulating hormone, adrenal corticotropin, melanocyte
stimulating hormone, parathyroid hormone, calcitonin, corticotropin
releasing factor, growth hormone releasing factor, thyrotropin releasing
factor, LH releasing factor, prolactin releasing factor, FSH releasing
factor, growth hormone release inhibiting factor, endorphins, enkaphalins,
vasopressin, oxytocin, proinsulin, insulin, glucagon, relaxin,
erythropoietin, secretin, gastrin, progastrin, angiotensin, neurotensin,
somatostatin, bombesin, caerulein and other growth effecting peptides. For
all hormones both the active form and the prohormone precursor forms are
included.
Among the enzymes of interest are the transmethylases, glycosidases,
esterases, peptidases, phosphatases, phosphokinases, phosphomutases,
sulfatases, sulfotransferases, mutarotases, acyl-coahydratases, fumarases,
aspartases, oleate hydratases, isomerases ketothiolases, aldolases and
carboxylases.
Of specific interest are the enzymes in the catagories of DNA restriction
endonucleases, exonucleases, proteinases, exopeptidases, thrombin and
plasmin. For all classes of enzymes both the active form and the zymogen
precursor forms are included.
Among the blood clotting factors of interest are
Factor XII (Wageman Factor)
Factor IX (Christmas Factor)
Factor VIII (Antihemophilic Factor)
and Factor X (Steamed Factor).
Among the antibodies and the antibody fragments are the gammaglobulin
classes IgG, IgA, IgM, IgE, and the F.sub.ab and F.sub.c fragments and the
constant regions of both heavy and light immunoglobulin chains.
Among the plasma proteins are included fibrinogen and plasminogen.
Among the structural proteins are included collagen, cell surface peptides
and cyto-skeletal peptides.
To produce the polypeptide of interest a synthetic or natural genes, either
constructed or isolated from cellular chromosones which code for
polypeptides, are modified by the covalent addition of a DNA fragment
containing DNA triplet codons directing the synthesis of a polyarginine
fragment. This hybrid DNA molecule made according to methods well known in
DNA recombinant technology results in the synthesis of a hybrid
polypeptide of interest with a polyarginine tail. The methods of this
invention are then used as a method of isolating the desired polypeptide.
The methods of this invention can be practiced in all cells, both
prokaryotic and eukaryotic, including bacteria, yeast, and eukaryotic
tissue culture.
EXAMPLE 1
STABILIZATION OF TRYP-E FUSED UROGASTRONE BY A C-TERMINAL POLY-ARG FUSION
The stabilization of urogastrone was demonstrated by the growth of E. coli
containing the urogastrone-polyarginine gene on plasmids, cell lysis
followed by assay for the presence of urogastrone. The following describes
the procedures used and the results.
Materials and Methods:
Carbenicillin.RTM., 3.beta.-indoleacrylic acid (IAA), tryptotphan and
thiamine were from Sigma London Ltd., Poole, Dorset, U.K. Case amino acids
were from DIFCO Inc., Detroit, Mich. U.S.A., all other chemicals were
analytical grade.
Growth of Cultures: Modified M9 medium (50 ml in 250 ml Nunc plastic flasks
(containing Na.sub.2 HPO.sub.4. 12H.sub.2 O (15.15 g/L), KH.sub.2 O.sub.4
(3.00 g/L), NH.sub.4 Cl (1.00 g/L), NaCl (0.5 g/L), Mg.sub.2
SO.sub.4.7H.sub.2 O (0.25 g/L), CaCl.sub.2.6H.sub.2 O (2.00 g/L), Case
amino acids (5.00 g/L), glucose (5.00 g/L), thiamine HCl (1 mg/L),
Carbenicillin Na salt (0.1 g/L) supplemented with Trp (40 .mu.g/ml) was
inoculated with E.coli from a glycerol stock and grown overnight on a New
Brunswick Orbital Incubator (18 h at 37.degree. C. and 150 rpm) to an
E.sub.600 of 4.3. This culture (5 ml) was used to inoculate modified M9
medium (250 ml in 800 ml plastic flasks) and incubation continued with
culture growth monitored by determining the E.sub.600.
Induction of Urogastrone: When the E.sub.600 of the culture reached 0.4,
IAA was added (20 .mu.g/ml). Samples (2 ml) were withdrawn for lysis and
assay of Urogastrone.
Lysis of E. coli: Sucrose buffer (60 .mu.l of 50 mM Tris/HCl, pH 7.4; 25%
w/v sucrose) was added to culture samples and incubated on ice for 5 min.
Additions of lysozyme (20 .mu.l of 10 mg/ml in PBS), EDTA (20 .mu.l of 500
mM in Tris/HCl, pH 8.0), and Triton X-100 (100 .mu.l of 0.6%) were made
with incubation on ice for five minutes between each. Samples were then
shaken vigorously at room temperature and stored at -20.degree. C.
Assay of Urogastrone: Lysed samples were thawed, centrifuged (12,000 g, 10
min, 4.degree. C.) and the supernatant (0.2 ml) diluted with PBS (1.4 ml).
Diluted samples were dialysed (4.degree. C.) against PBS (three changes
over 24 h) then stored at -20.degree. C. Samples were thawed and assayed
using a radio-receptor assay (Hollenberg et al., (1980) Mol. Pharmacology.
17, 314-320). Urogastrone receptor activity is expressed as the activity
equivalent to a known weight of purified human .beta.-urogastrone standard
(uro.equivs.).
Results and Discussion:
Cultures of E.coli HB101 containing the plasmids PWT221-Uro and the plasmid
coding for polyarginine tailed urogastrone, PWT221-Uro-polyarginine, gave
similar growth profiles. However, the activity of PWT221-Uro-polyarginine
continued to increase throughout the growth cycle whereas PWT221-Uro did
not. Furthermore, at the end of the experiment, the activity of
PWT221-Uro-polyarginine culture was 3.times. greater than PWT221-Uro (FIG.
1). The use of modified M8 medium which is low in Trp, and the addition of
IAA, the Tryp gene inducer, ensures that the urogastrone gene is being
transcribed at its maximum rate. Therefore, if a stable protein is being
produced, it should be translated from the mRNA at a constant rate and
accumulate throughout the culture's growth. The evidence presented in FIG.
1 indicates that the polyarginine tail is stabilizing the expression and
therefore increasing the amount of urogastrone produced by this culture.
This may be the result of interactions with anionic polymers associated
with the insoluble cell membrane fraction. Such an association may reduce
the accessibility of the protein to soluble proteolytic enzymes produced
by E.coli.
FIG. 1 illustrates the expression of the urogastrone gene in E. coli with
and without a polyarginine tail. E. coli HB101 containing the plasmids
PWT221-Uro and PWT221-Uro-polyarginine were cultured and induced with IAA.
Growth and urogastrone activity were measured in lysates as described in
Methods. Growth (O) and urogastrone activity (.quadrature.) of PWT221-Uro;
growth () and urogastrone activity () of PWT221-Uro-polyarginine.
EXAMPLE 2
PURIFICATION OF TRYP-E FUSED UROGASTRONE USING THE PROPERTIES OF A
POLYARGININE TAIL
The purification of urogastrone utilizing the polyarginine tail was
performed on both a small scale (800 ml E. coli culture) and a larger
scale (32L E. coli culture). The results are shown in Tables 1 and 2. The
procedure used was the following.
Materials and Methods:
Carboxypeptidase B (CPB) Type 1 DFP (180 units/mg) was obtained from Sigma;
SP-Sephadex C-25.RTM. and CnBr Sepharose were obtained from Pharmacia Fine
Chemicals AB, Uppsala 1, Sweden, Spectrapor.RTM. dialysis tubing (cut off
3500) was obtained from Spectrum Medical Industries, Los Angeles, U.S.A.
Commassie Blue R250 was from Serva GMBH & Co, Heidelberg 1, W. Germany.
Biorad.RTM. protein assay reagent was from Bio-Rad Lab., 8000 Munich 66,
West Germany. Pyrotell limulus amoebocyte lysate assay for endotoxins was
from Associates of Cape Cod Ltd., Mass., U.S.A. All other chemicals were
analytical reagent grade. The 7L fermenter was supplied by LH Engineering
Ltd, Stoke Poges, U.K., and the 14L fermenter was supplied by Chemapec
Ltd, Abingdon, Oxon, U.K. AVP homogeniser was from AVP Co. Ltd., Crawley,
Suffex, U.K.). The sonicator was from Ultrasonics Ltd, U.K.
Fermentation: Shake flask cultures, grown on Modified M9 medium as
described in Example 1 were used to inoculate (at 4-10% v/v) either 5L of
medium in a 7L fermenter, (stirred at 750 rpm) or 8L of medium with
additional glucose (15 g/L) and Case amino acids (15 g/L) in 14 L
fermenters (stirred at 550 rpm). All fermentations were at 37.degree. C.,
pH 7.0, with an aeration rate of one v/v per min.
Urogastrone extraction: Cells were harvested from cultures by
centrifugation (4,000 g, 20 mins, 20.degree. C.) and disrupted in lysis
buffer (5M urea, 40 mM Tris-acetate/NaOH pH 9.5) either by a sonicator (30
ml aliquots with a 5 mm probe, 50 W, on ice for 10 min) or by an AVP
homogenizer (8,000 psi, 2 passes) and centrifuged (16,000 g, 30 mins,
20.degree. C.). The supernatant was adjusted to pH 5.5 with 1N HCl and
centrifuged (16,000 g for 60 mins, 20.degree. C.). This supernatant was
loaded onto SP-Sephadex.
SP-Sephadex chromatography: SP-Sephadex C-25 was equilibrated in Tris/urea
(5M urea, 40 mM Tris-acetate pH 5.5). All chromatography was performed at
room temperature. The flow rate for loading, washing and elution was
constant at one bed volume/10 min.
Preparation of CPB-Sepharose: CPB (20 mgs in 10 mls of 0.1M NaH.sub.2
CO.sub.3, pH 8.3) was added to 10 mls of CnBr Sepharose as described by
Pharmacia (Pharmica, in Handbook on Affinity Chromotography, p 14-18,
Uppsala, 1, Sweden.) and reacted for 16 hours at 4.degree. C. Essentially
all the protein was coupled to the gel with a 50% recovery of CPB
activity. CPB-Sepharose was stable at 4.degree. C. for at least 3 months
if stored in phosphate buffered saline (PBS) and azide (0.1%).
CPB digestion: The first SP-Sephadex eluate was digested with CPB-Sepharose
by gentle end over end rotation. CPB-Sepharose was removed from digested
urogastrone by filtration on a sintered glass funnel.
Gel Analysis: Polyacrylamide gels were prepared by the method of Ornstein
and Davis, (Ornstein, L. (1964) Ann. N.Y. Acad. Aci. 121,321-349 and
Davis, B. J. (1964) Ann. N.Y. Acad. Aci. 121,404-427). SDS-polyacrylamide
linear gradient gels were prepared by the method of Laemmli, (Laemmli, U.
K. (1970) Nature (London) 277,680). Gels were fixed and stained using
Coomassie Blue (2.5 g/L of methanol:acetic acid:water 3:1:6) and destained
in this same solvent mix.
Assays: Bio-Rad dye reagent was used for all protein determinations with
BSA as standard. Carboxypeptidase B was assayed using the method of Corbin
et al. (Corbin, N. C., Hugli, T. E. and Muller-Eberhard, H. J. (1976)
Anal. Biochem. 73,41-51.). Endotoxins were assayed using Pyrogel using the
suppliers recommendations. Dry weight was determined after freeze drying.
All other methods are as described in Example 1.
Results and Discussion:
Using a two step ion-exchange process (table 1), the urogastrone from an
800 ml culture was purified to a single band on polyacrylamide gel
electrophoresis (PAGE). This purification was based on the loss of
positive charge caused by removal of the polyarginine tail with
CPB-Sepharose. This change in charge was indicated by the reduction in
salt required to elute urogastrone from SAP-Sephadex after CPB digestion
(30 mM as opposed to 120 mM). This small scale process was successfully
scaled up 40 fold to yield 64 mgs of purified urogastrone with a good
yield (table 2). This preparation has been analysed by gel electrophoresis
(single band on SDS-PAGE and PAGE), assayed for endotoxins (1 ng
pyrogen/mg protein), and the dry weight determined (1.3 mgs/mg protein).
The Small Scale Purification of Urogastrone was performed as follows with
the results shown in Table 1. The crude load was from an 800 ml E.coli
culture grown in a 7 L fermenter to an E.sub.600 of 2.2 and lysed by
sonicaton. The SP-Sephadex column (10 mm.times.20 mm) was washed with 10
mls of Tris/urea and eluted with a linear gradient (0-300 mM NaCl in
Tris/urea). Fractions containing peak Urogastrone activity were pooled,
adjusted to pH 8.1 with 1M NaOH and digested with 100 .mu.L of
CPB-Sepharose for 2 hours at 22.degree. C., pH 8.1. The CPB treated
urogastrone was dialyzed overnight at 4.degree. C. against 8 L of 40 mM
Tris/acetate, pH 5.5; then urea was added to 5M and the CPB digested load
was pumped onto a SP-Sephadex column (10 mm.times.10 mm) in Tris/urea.
After washing with 5 mls of Tris/urea, the urogastrone was eluted with a
linear gradient (0-125 mM NaCl in Tris/urea) and the urogastrone activity
pooled.
TABLE 1
______________________________________
SMALL SCALE PURIFICATION OF UROGASTRONE
Purifi-
Volume Protein Urogastrone cation
Sample (ml) (mg) (mg equivs)
yield Factor
______________________________________
SP-Sephadex
Column 1
Crude load
21 121 4.4 (100%) (1)
Non-adsorbed
26 83 0.3 8% --
and wash
Elution pool
6.4 3.6 2.4 55% 19
SP-Sephadex
Column 2
CPB digested
6.4 3.6 2.3 53% 19
load
Non-adsorbed
12 0 0 0 --
and wash
Elution pool
5.5 1.2 1.7 39% 51
______________________________________
The Large Scale Purification of Ur | | |
