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BACKGROUND AND PRIOR ART
In the mid-1970's, it was discovered that opium alkaloids such as morphine
and heroin bind to specific receptors in brain and other tissues. The
specific binding reaction was found to be prerequisite to the production
of the characteristic biological effects of analgesia and euphoria for
which the opiates are well known. The findings led to the discovery of
endogenous substances which also bind to the opiate receptors. These
naturally occuring compounds are termed endorphins, for endogenous
morphine-like substances. A number of such compounds have been isolated
and characterized and shown to be peptides having similarities in their
amino acid sequence. The most active of these substances is
.beta.-endorphin, a polypeptide of 31 amino acids whose sequence (except
for the C-terminal gistamine) is shown in FIG. 1.
In the mammaliam pituitary, .beta.-endorphin is synthesized as a precursor
protein of molecular weight about 30,000. The precursor protein includes
the amino acid sequences of adrenocorticotropic hormone (ACTH),
.beta.-lipotropin (.beta.-LPH), .alpha.-melanocyte stimulating hormone
(.alpha.-MSH), .beta.-melanocyte stimulating hormone (.beta.-MSH),
corticotrophin-like intermediate lobe peptide (CLIP) met-enkephalin and
.beta.-endorphin. (See, e.g. Roberts, J. L. and Herbert, E., Proc. Natl.
Acad. Sci. U.S.A. 74, 4826 (1977); Mains, E. and Eipper, B. A., J. Biol.
Chem. 251, 4115 (1976); Nakanishi, S., et al., Proc. Natl. Acad. Sci. 73,
4319 (1976)). Normally, the precursor protein is processed by
post-translational proteolysis and glycosylation, to generate the
individual peptide hormones (Roberts, J. L., et al., Biochemistry 17, 3609
(1978); Eipper, B. A. and Mains, R. E., J. Biol. Chem. 253, 5732 (1978)).
To the extent the functions of te hormonal peptides contained within the
precursor protein sequence are understood, they appear to be related
generally to physical and biochemical adaptations to stress. The
physiological responses to .beta.-endorphin appear to be analogous to the
physiological effects of such opium alkaloids as morphine and heroin. Such
effects are well known and include analgesia, altered emotional state, and
reduced anxiety.
The medical uses of .beta.-endorphin are generally those for which the
opiate alkaloids are presently employed. Therefore, the availability of an
endogenous substance capable of producing the same effects is highly
attractive to the medical profession. In addition, the expansion of
medical uses for the opiates coupled with world population increases will
lead to a projected world-wide shortage of morphine and its analogs within
a few years. At the present time, .beta.-endorphin is known to be useful
as an analgesic and in the treatment of intractable pain. Intra-cerebral
administration of .beta.-endorphin, via an implanted delivery tube, has
been employed in the successful treatment of such forms of intractable
pain as phantom limb pain. In addition, .beta.-endorphin is effective as
an intravenously administered analgesic. .beta.-endorphin has been
reported to be effective in the treatment of certain mental disorders
related to mood, affect and anxiety, including schizophrenia. It is also
known that .beta.-endorphin binds to the opiate receptors in the gut so
that it can be applied in the treatment of such gastrointestinal disorders
as severe diarrhea, which presently are treated with opiates. It has also
been shown that the adminstration of .beta.-endorphin induces increased
plasma levels of prolactin and may therefore prove effective as a
long-term birth control agent. Because of the presently known and
predicted uses for .beta.-endorphin in medical care, there is presently a
substantial market for the compound as an investigational drug. Prior to
the present invention, the compound was obtainable in small quantity and
great expense either by purification from brain extracts or by chemical
synthesis. At current prices, the market for .beta.-endorphin as an
investigational drug in the United States alone is estimated at several
million dollars per year. Clearly, a method for producing .beta.-endorphin
in quantity, with substantial economies of scale, is highly desirable. The
present invention provides such a method.
Although .beta.-endorphin can be obtained from extracts of brain tissue,
the yields are relatively small. Chemical synthesis has been achieved by
Li, C. H., et al., Biochem. Biophys. Res. Commun. 71, 19 (1976). DNA
sequences coding for all or part of the ACTH/.beta.-endorphin precursor
protein have been cloned by the cDNA method (Roberts, J. L., et al., Proc.
Natl. Acad. Sci. 76, 2153 (1979); Nakanishi, S., et al., Nature 278, 423
(1979)). The cloning of a DNA coding for a portion of the
ACTH/.beta.-endorphin precursor protein is described in U.S. Pat. No.
4,322,499 incorporated herein by reference. The genetic material cloned
therein was employed as a starting material in the present invention.
Methods for the expression of heterologous DNA in a microorganism are now
known. In principle, the heterologous DNA coding sequence is inserted in a
DNA transfer vector at a point located within an expressible operon. The
inserted sequence must be in reding frame phase with the coding sequence
of the operon, and oriented in the same direction with respect to
translation. When the conditions are met, translation of the operon
results in "readthrough" to the inserted coding sequence such that the
protein produced is a fusion protein comprising an N-terminal amino acid
sequence coded by the expressible operon, followed by an amino acid
sequence coded by the insert. See Polisky, B., et al, Proc. Natl. Acad.
Sci., 73, 3900 (1976); Itkura, K., et al, Science, 198, 1056 (1977).
Several expressible operons have been employed, including insertion in the
.beta.-galactosidase gene, the .beta.-lactamase gene, and the tryptophan
operon.
Although the genetic code is said to be "universal" in the sense that all
known living organisms use the smae code, it is well known that higher
organisms, such as mammals, preferentially employ a set of condons which
differs from that preferred my microorganisms such as bacteria. This
observation has led investigators to choose synthetic coding sequences
employing codons preferred by bacteria. Such a strategy does not appear to
be necessary, at least for the expression of immunologically
cross-reactive material, since both synthetic and naturally occuring
sequence can be expressed in bacteria to yield an immunologically active
product (Itakura, K., et al., Science 198, 1056 (1977); Martial, J. A., et
al., Science 205, 602 (1979)). Until the present invention, however,
evidence of a biologically functional hormone has not been presented,
either for synthetic or naturally occuring coding sequences. The present
invention demonstrates the feasibility of using coding sequences
comprising naturally occurring mammalian codons to achieve expression of
biologically active protein by a recombinant microorganism.
SUMMARY OF THE INVENTION
The present invention is believed to provide the first instance of the
synthesis of a mammalian hormone by a microorganism transformed by a
coding sequence comprising naturally occurring mammalian codons, wherein
the biological activity of the product was demonstrated. The invention is
exemplified by the bacterial synthesis of mouse .beta.-endorphin. The
mouse endorphin differs from human endorphin merely by two amino acids and
the mouse endorphin is thought to be biologically active in humans.
The microbiological synthesis of .beta.-endorphin is accomplished in the
present invention by modifying an existing cloned coding sequence,
expressing the modified coding sequence as a fusion protein in a
microorganism, modifying the fusion protein to specifically remove the
non-endorphin portion of the fusion protein, yielding the .beta.-endorphin
sequence per se, purifying .beta.-endorphin from a bacterial lysate and
proving the identity of the purified material by immunological reactivity
and by specific tests of biological function.
Abbreviations used herein are as follows:
______________________________________
A = Adenine dATP = deoxyadenosine triphosphate
G = Guanine dGTP = deoxyguanosine triphosphate
C = Cytosine
dCTP = deoxycytosine triphosphate
T = Thymine dTTP = thymidine triphosphate
______________________________________
(DNA base sequences are written as single strands with the indicated
polarity, e.g. 5'-ATGC-3'. When both strands are shown, the lower strand
has the opposite polarity. It will be understood that deoxynucleosides are
intended in all DNA sequences).
______________________________________
Ala = Alanine Leu = Leucine
Met = Methionine Ile = Isoleucine
Ser = Serine Val = Valine
Gly = Glycine Lys = Lysine
Cys = Cysteine Arg = Arginine
Glu = Glutamic acid
Phe = Phenylalanine
Asp = Aspartic acid
Tyr = Tyrosine
Gln = Glutamine Trp = Tryptophan
Asn = Asparagine His = Histidine
Thr = Threonine Pro = Proline
______________________________________
Amino acid sequences are written from the NH.sub.2 -terminal end on the
left to the COOH-terminal end on the right.
HEPES=N-2-hydroxyethylpiperatine-N'-2ethanesulfonic acid
EDTA=Ethylenediaminetetracetic acid
Tris=Hydroxmethylaminomethane (base) or hydroxymethyl
aminomethanehydrochloride (acid)
SDS=Sodium dodecyl sulfate
.beta.-gal=.beta.-galactosidase
DETAILED DESCRIPTION OF THE INVENTION
The cloned coding sequence used as the starting point in the present
invention contained the coding information for amino acids 44-90 of the
beta lipotropin portion of the ACTH/.beta.-endorphin precursor protein. A
map of the precursor protein and detailed structure of a portion of the
cloned coding sequence are shown in FIG. 1. The sequence codes for all of
.beta.-endorphin except the C-terminal glutamine. The strategy of the
present invention involves insertion of the mammalian coding sequence into
an internal position in a bacterial gene such that the cloned mammalian
sequence is in phase with the bacterial coding sequence. It is thus
necessary to modify the cloned mammalian sequence fragment in order to
re-create the codon for the carboxy-terminal amino acid, insert a stop
codon and link the fragment in phase to a bacterial gene. It is also
necessary to devise a method for the release of mature .beta.-endorphin
from the fusion protein.
The approach to these problems is outlined in FIG. 2. The cloned mammalian
coding fragment was released from the DNA transfer vector in which it was
inserted by HindIII endonuclease digestion, since the fragment had been
inserted into the transfer vector using HindIII linkers. The released
fragment was first cleaved in the .beta.-MSH coding region with HpaII
endonuclease in order to facilitate the insertion in phase with respect to
reading frame, in the bacterial gene. The single stranded HpaII and
HindIII terminae were then partially filled in by reaction catalysed by
reverse transcriptase in the presence of dATP and dCTP. The reaction
re-created the 3'-terminal glutamine codon and added a cytosine residue at
the HpaII site. The remaining unpaired terminal nucleotides were then
removed by Sl nuclease digestion and the blunt-ended fragment connected to
a synthetic octanucleotide containing the recognition site for EcoRl, in a
DNA-ligase catalysed reaction.
In principle, the cloned mammalian coding sequence fragment could be
inserted into a variety of bacterial genes including but not limited to
the .beta.-galactosidase gene, the .beta.-lactamase gene, and genes of the
tryptophan operon. The partial filling in of unpaired single stranded ends
can be employed to advantage in many similar situations. The choice of
bacterial gene into which to insert the coding sequence fragment will
depend upon the reading frame phase of the insertion site, the
availability of suitable restriction sites and desired properties of the
resulting fusion protein. The present invention is exemplified by an
insertion into the bacterial gene coding for .beta.-galactosidase, at an
EcoRl site which is known to occur at the codon for amino acid 1004
(Itakura, K., et al., Science 198, 1056 (1977)).
The use of the synthetic octanucleotide containing the recognition site for
EcoRl served a dual purpose of providing the EcoRl terminae necessary for
ligation with the .beta.-galactosidase gene and also provided a stop codon
immediately following the 3'-terminal glutamine codon. The modified
fragment was then inserted into the EcoRl site of a DNA transfer vector
carrying the appropriate bacterial gene. For example, in the present
invention, the plasmid pBR322 carrying the lac control region and the
coding sequence for .beta.-galactosidase was employed. The plasmid has a
single EcoRl site at the codon for amino acid 1004 of
.beta.-galactosidase, and is designated p.beta.gal.
The recombinant plasmid formed by inserting the modified .beta.-endorphin
coding sequence at the EcoRl site designated p.beta.gal-end, was used to
transform a culture of the bacteria Escherichia coli. Growth of the
transformed cells in culture resulted in the formation of a fusion protein
under control of the lac promotor. Synthesis of the fusion protein could
be demonstrated by SDS-polyacrylamide gel electrophoresis of cell proteins
(FIG. 3). Extracts of cells carrying the plasmid p.beta.gal-end lacked a
band in the position expected for the normal .beta.-galactosidase protein,
but demonstrated the appearance of a new band reflecting a protein
approxmately 30 amino acids larger than .beta.-galactosidase, the size
expected for the .beta.-galactosidase-.beta.-MSH-.beta.-endorphin fusion
protein. It was found that the fusion protein was soluble and could be
recovered from a high-speed pellet of a cell sonicate, where it
represented a substantial proportion of the recoverable protein.
The steps used to release mature .beta.-endorphin from the hybrid protein
are shown in FIG. 4. The arginine residue preceding the .beta.-endorphin
sequence (FIG. 1) was used as a site for proteolytic cleavage by trypsin.
Since .beta.-endorphin contains several internal lysine residues that
would be susceptible to trypsin cleavage, it was necessary to specifically
protect the lysine residues against trypsin attack. It was discovered that
the lysine residues in .beta.-endorphin could be protected from attack by
trypsin by prior reaction with citraconic (methylmaleic) anyhdride in
vitro (Dixon, H. B. F. and Perham, R. N., Biochem. J. 109, 312 (1968)).
Thus, after dissolving the precipitated fusion protein and treating with
citraconic anhydride at pH 9, the .beta.-endorphin (containing the
modified lysine groups) was released from the fusion protein by cleavage
with trypsin. Native .beta.-endorphin was subsequently produced by
removing the citraconic groups at pH 3. The native .beta.-endorphin was
then further purified from this preparation as described infra.
The .beta.-endorphin was shown to be immunologically reactive with
antiserum raised against mouse endorphin. By quantitative analysis of the
results, it was estimated that the bacteria synthesized approximately
8.times.10.sup.4 molecules of .beta.-endorphin per cell. The
.beta.-endorphin prepared as described was further characterized for
biological function in two separate tests. The first test was the ability
of the endorphin to bind to opiate receptors from rat brain and to compete
in such binding with a known opiate agonist (enkephalinamide) and a known
opiate antagonist (naloxone). Endorphin, prepared according to the
invention, displaced both the opiate agonist and the opiate antagonist
from the rat brain opiate receptors.
In a separate assay, the ability of endorphin, prepared according to the
invention, to inhibit the stimulation of adenylate cyclase activity by
prostaglandin E.sub.1 in a neuroblastomaglioma hybrid cell line was
tested. The prostaglandin E.sub.1 stimulation is known to be inhibited by
morphine and the inhibitory effect of morphine is reversed by naloxone.
Endorphin produced according to the invention showed a significant
inhibition of prostaglandin E.sub.1 stimulation of adenylate cyclase
activity, which inhibition was reversed by addition of the opiate
antagonist naloxone.
The present invention represents the first known instance of the synthesis
of a mammalian hormone by a microorganism, transformed with a coding
sequence comprising naturally occurring mammalian codons wherein proof of
a functional protein product was adduced. The techniques disclosed herein
may be applied to the preparation of peptides that differ from the mouse
endorphin demonstrated in the examples. Thus, human endorphin differs from
the mouse peptide only at position 27 where a tyrosine residue is present
instead of a histidine and at position 31 where a glutamine residue
replaces a glutamie acid. The mouse endorphin is known to cross-react
immunologically with human endorphin and no difference in biological
activity in human cells is known. The .beta.-endorphin expression plasmid
described herein may be used as starting material to program bacterial
plasmids that direct the synthesis of analogs of .beta.-endorphin which
may have altered or selectively different opiate agonist or antagonist
activities. In particular, modifications of the C-terminal part of the
amino acid sequence are known to profoundly affect biological activity.
Li, C. H., et al, Proc. Natl. Acad. Sci. 76, 3276 (1979), have shown that
Gly.sup.31 -endorphin, Gly.sup.31 -endorphinamide and Gly.sup.31
-endorphinylglycine are more potent opiate agonists than endorphin itself.
The present invention provides methods for modifying the naturally
occurring sequence, as originally cloned, so that such modified amino acid
sequences are expressed. Furthermore, it is anticipated that other
feasible modifications within the scope of the invention, will yield
endorphin-like peptides that function as opiate antagonists.
The steps in constructing a system for synthesizing .beta.-endorphin using
bacteria are next described in detail, with particular reference to the
synthesis of mouse endorphin, as described in the following examples.
The characteristic features of the described method are applicable to the
synthesis of other endorphins from other species and in particular to the
synthesis of human .beta.-endorphin.
EXAMPLE 1
Construction of a Plasmid for the Expression of .beta.-endorphin Sequences
by E. coli
In the following experiments, digestions with restriction endonucleases
were carried out under conditions optimized for each enzyme. Enzymes were
obtained commercially (New England BioLabs, Cambridge, Mass.) and optimal
reaction conditions according to the supplier's recommendations were
employed unless noted otherwise. The use of reverse transcriptase and
suitable reaction conditions have been described previously by Seeburg,
P.H., et al., Nature 276, 795 (1978); Seeburg, P. H., et al., Nature 270,
486 (1977); and Shine, J., et al., Nature 270, 494 (1977). Recombinant
plasmids were isolated from chloramphenicol-amplified cultures as
previously described by Bolivar, F., et al., Gene 2, 95 (1977).
A mammalian coding sequence of 150 base pairs was obtained from the plasmid
pMAE-1 (Roberts, J. L., et al., supra.) by HindIII endonuclease digestion,
followed by electrophoresis in a 7% (w/v) polyacrylamide gel. The
mammalian DNA coding fragment was then modified by digestion of .about.500
ng DNA with five units of HpaII endonuclease at 37.degree. C. for 1 hour
in 20 .mu.l of 6 mM Tris pH 7.5, 6 mM MgCl.sub.2 and 6 mM
.beta.-mercaptoethanol. The reaction mixture was then extracted with
phenol and DNA was recovered by ethanol precipitation. The DNA was then
redissolved in 50 .mu.l of a reaction mixture containing reverse
transcriptase buffer and 500 .mu.M each of dATP and dCTP. The mixture was
incubated at 37.degree. with 10 units of reverse transcriptase for 15
minutes. At the end of the reaction, unincorporated triphosphates were
removed by chromatography on Sephadex G-50 (TM Pharmacia Inc., Uppsala,
Sweden). DNA was recovered by ethanol precipitation. The DNA was then
re-dissolved in 50 .mu.l of 0.3 M NaCl, 30 mM Na acetate pH 4.5, 3 mM
ZnCl.sub.2, incubated with 5 units of S.sub.1 nuclease at 20.degree. C.
for 5 minutes, then recovered by phenol extraction and Sephadex G-50
chromatography.
The double-stranded blunt-ended DNA was then covalently joined to the
synthetic octamer
##STR1##
(Heyneker H. L. stal, Nature, 263 748(1976). The octamer functions as a
linker sequence and contains the restriction site for endonuclease EcoRl.
See Scheller, R. H., et al., Science 196, 177 (1977). The joining reaction
was catalysed by bacteriophage T.sub.4 DNA ligase (New England BioLabs) in
a 40 .mu.l reaction mixture containing 60 mM Tris (pH 7.5), 8 mM
MgCl.sub.2, 10 mM .beta.-mercaptoethanol, 1 mM ATP, 2 .mu.l of enzyme,
.about.500 ng DNA and a 20-fold molar excess of synthetic linker. The
reaction mixture was incubated for 24 hours at 4.degree. C. After the
addition of 0.1 M NaCl and digestion with 50 units of EcoRl endonuclease
at 37.degree. C. for 5 hours, the unreacted linker fragments were removed
by Sephadex G-50 chromatography.
The DNA transfer vector chosen for insertion was the plasmid p.beta.gal
described supra. The plasmid was digested with EcoRl endonuclease to
convert the circular plasmid DNA to linear DNA having a unique sequence.
The linear DNA was further treated with alkaline phosphatase to prevent
the subsequent formation of plasmid p.beta.gal in a DNA ligase catalysed
reaction, as described in U.S. Pat. No. 4,264,731, incorporated herein by
reference. The modified mammalian DNA coding sequence was inserted into
the treated p.beta.gal DNA in a DNA ligase catalysed reaction, as
described in the references cited at the beginning of this example.
Recombinant plasmids were used to transform E. coli RRl, Bolivar, F.,
supra. Single colonies of recombinant transformants were isolated and
grown in 3 ml cultures to prepare plasmid DNA therefrom. The mammalian
insert contains an asymmetrically located HaeIII site (GGCC) which yields
differently sized fragments upon HaeIII endonuclease digestion, depending
upon the orientation of the insert. A clone having a correctly inserted
mammalian sequence was selected for further analysis. The nucleotide
sequence of the inserted DNA was determined by the method of Maxam, A. and
Gilbert, W., Proc. Natl. Acad. Sci. 74, 560 (1977). The sequence of the
modified insert was shown to be identical to that shown in FIG. 2.
EXAMPLE 2
Expression of a .beta.-Galactosidase-.beta.-Endorphin Fusion Protein
E. coli RRl cells harboring either plasmid p.beta.gal-end or p.beta.gal
were grown at 37.degree. C. to mid-log phase in L-broth and harvested by
centrifugation. The cells were dissolved in SDS sample buffer (Laemmli,
U.K., Nature 227, 680 (1970)). Protein equivalent to 50 .mu.l of original
culture was subjected to electrophoresis in a 7% (w/v) polyacrylamide gel
and visualized by staining with coomassie blue. Results are shown in FIG.
3. Lane a shows the protein banding pattern derived from E. coli RRl; Lane
b shows proteins obtained from E. coli RRl induced with 2 mM
isopropyl-.beta.-thiogalactoside (IPTG); Lane c: proteins obtained from E.
coli RRl carrying p.beta.gal; Lane d: proteins obtained from E. Coli RRl
carrying p.beta.gal and induced with IPTG; Lane e: proteins obtained from
E. coli RRl carrying p.beta.gal-end; Lane f: proteins from E. coli RRl
carrying p.beta.gal-end induced with IPTG; Lane g: protein obtained from
E. coli RRl carrying p.beta.gal-end prepared by harvesting the cells by
centrifugation, resuspending the cells in phosphate buffered saline,
disrupting the cells by sonication and fractionating the sonicate by
centrifugation at 15,000 rpm for 30 minutes and analyzing the supernatant
fraction; Lane R: protein from E. coli RRl carrying p.beta.gal-end
fractionated as described in Lane g, analyzing the pellet after
solubilization in SDS: Lane i: proteins obtained from E. coli RRl carrying
p.beta.gal-end after treatment to amplify the plasmid DNA with 50 .mu.g/ml
chloramphenicol, followed by 3 hours' growth in fresh L-broth.
The electrophoresis banding pattern of proteins derived from cells
transformed with p.beta.gal-end shows substantial amounts of protein not
found in non-recombinant cultures and decreased amount of
.beta.-galactosidase. The position of .beta.-galactosidase is shown by
arrow #1 and that of the .beta.-galactosidase-.beta.-endorphin fusion
protein by arrow #2. When cells are disrupted by sonication, the fusion
protein is found associated with the cell pellet. Increased amounts of the
fusion protein are formed after induction with IPTG, consistent with the
conclusion that expression of the .beta.-galactosidase-.beta.-endorphin
fusion protein is under lac operon control. E. coli RRl carrying
pbeta-gal-end, and the plasmid pbeta-gal-end, were placed on deposit at
the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.
on Mar. 6, 1980. The bacteria has ATCC accession number 31614. The plasmid
has ATCC number 40024.
EXAMPLE 3
Release of .beta.-endorphin from the Fusion Protein
The sequence of steps detailed in this example is shown diagrammatically in
FIG. 4. The pellet fraction, obtained as described in Example 2, from
cells bearing p.beta.gal-end, was dissolved in 10 ml of 6 M guanidinium
chloride, 1% (v/v) 2-mercaptoethanol and centrifuged at 20,000 rpm for 1
hour. Citraconic anhydride (obtained from Fisher Chemical Company, Santa
Clara, California) was added to the supernatant fraction in three lots, 10
.mu.l each, over a 15-minute period, during which time the pH was
maintained between 9 and 11 by the addition of 1 N NaOH, as described by
Dixon, H. B. F. and Perham, R. N., supra. The reaction mixture was then
dialyzed overnight against 50 mM ammonium bicarbonate.
The dialyzed solution was incubated with trypsin (Worthington Biochemical
Corporation, Freehold, N.J.), 0.5 mg/ml, at 37.degree. C. for 12 hours.
Trypsin was then inactivated by addition of phenylmethylsulfonyl fluoride
(PMSF) to 1 mM for a 1-hour additional incubation period. The protein was
deprotected by the addition of formic acid to 1% (v/v) and lyophilised.
The dried protein was re-dissolved in Tris buffer pH 7.6 to yield a final
protein concentration of 0.5-1.0 .mu.g/.mu.l. The foregoing preparation
was used for some of the immunological and biological activity tests.
This procedure has general utility for cleavage of a leader or prepeptide
sequence, since such sequences are often found to be joined by a lys-arg
connection to the active peptide. The procedure will also be effective in
cases where internal arginine residues exist in the active peptide, since
those arginines will often be protected by the folded configuration of the
peptide.
EXAMPLE 4
Purification of .beta.-endorphin
Further purification of .beta.-endorphin was accomplished by a modification
of the technique devised for extraction and purification of ACTH by Rees,
L. H. et al., Endocrinology 87, 254 (1971). To 0.5 ml aliquots of the
endorphin preparation described in Example 3, were added 0.5 ml of horse
serum (Grand Island Biological Company, Santa Clara, Ca.) and 50 mg glass
powder, 140 mesh (Corning Glass Works Leached Silica Glass, Code No. 7930,
Corning, N.Y., water washed to remove fines and oven dried 24 hours at
120.degree. C.) in a 15 ml plastic centrifuge tube. These samples were
vortexed for 30 seconds and then centrifuged for 5 minutes at 2,000 rpm.
The supernatant was discarded and the glass adsorbant was washed with 3 ml
of water. The endorphin was removed from the glass by adding 1 ml of 50%
(v/v) acetone in 0.25-0.5 N HCl, vortexing for 30 seconds and then
centrifuging as described. The supernatant was then transferred to
polystyrene tubes and evaporated to dryness with a fine stream of nitrogen
in a water bath at 45.degree. C. Dried sample were re-dissolved in Tris
buffer pH 7.6.
EXAMPLE 5
Immunological Activity
Immunological activity of .beta.-endorphin produced according to the
present invention was measured by its ability to compete with
radioactively labeled human .beta.-endorphin for antiserum directed
against mouse .beta.-endorphin. The human hormone differs from that of
mouse only at position 27, where a tyrosine is present instead of
histidine residue. This change does not inhibit the ability of the human
hormone to react to antiserum raised against the mouse hormone. Antiserum
to mouse .beta.-endorphin was prepared as described by Allen, et al.,
Proc. Nat'l. Acad. Sci. 75, 4972 (1978). Human .beta.-endorphin was
synthesized by the solid phase technique as described by Li, et al. supra,
and was radioactively labeled with .sup.125 I by a chloramine-T procedure
(Hunter H. M. and Greenwood, J. C. Native, 194, 495 (1962). The iodinated
peptide was purified by absorption to 35 mg glass powder in 10 ml of
buffer (ph 7.4) containing 0.05 M sodium phosphate, 0.25% human albumin
and 0.5% .beta.-mercaptoethanol. After mixing for 5 min., the suspension
was spun at 3500 rpm for 5 min., washed once with 5 ml water and then
eluted with 2 ml of 40% (v/v) acetone in 0.25 N HCl.
In the assay, a dilution series of .beta.-endorphin sample prepared
according to the present invention was incubated with a fixed amount of
labeled human endorphin and a fixed amount of antiserum. Reaction mixtures
contained 75 .mu.l of .sup.125 I-human .beta.-endorphin (10,000) cpm), 50
.mu.l of protein solution prepared as described in Example 4 from either
p.beta.gal-containing bacteria or from p.beta.gal-end containing bacteria,
in 0.05 M sodium phosphate pH 7.4, 0.25% (w/v) human albumin and 0.5%
.beta.-mercaptoethanol. Antiserum was added to a final volume in the
reaction mixture of 150 .mu.l. Incubations were carried out at 4.degree.
C. for 24 hours. Antigen-antibody complexes were separated from the
reaction mixture by adsorption to a dextran-charcoal suspension containing
3% (w/v) charcoal (Norit A, Pfanstiehl Chemical Company, Waukegan, Ill.),
0.75% (w/v) dextran (Schwartz-Mann, Van Nuys, Calif.) and 60% (w/v) horse
serum (Grand Island Biological Company, Santa Clara, Calif.) in 0.05 M
sodium phosphate pH 7.6. The suspension was incubated for 5 minutes at
4.degree. C., then centrifuged at 2,500 rpm for 15 minutes. The
supernatant containing bound antigen was aspirated with a glass bent-tip
pipette. The percentage of radioactivity remaining in the pellet was
compared to the total radioactivity determined prior to incubation and
plotted as a function of the concentration of the cell extracts. The
results are shown in FIG. 5.
It is apparent from FIG. 5 that extracts derived from cells bearing plasmid
p.beta.gal-end (solid circles) competed with labeled human
.beta.-endorphin for the available antibody. In a control experiment,
extracts from cells carrying the plasmid p.beta.gal (triangles) had no
competitive effect. The coefficients of variation in these experiments
were less than 5%.
EXAMPLE 6
Receptor Binding Activity
In the following experiment, the ability of .beta.-endorphin, made
according to the present invention, to competitively inhibit the binding
of a radioactively labeled opiate agonist or a labeled antagonist for the
binding to rat brain opiate receptors was tested. The opiate receptor
preparation was prepared from rat membrane preparation isolated from male
Sprague-Dawley rats (200-220 g) killed by decapitation. The brains, except
for the cerebellum, were homogenized in 40 volumes of ice-cold Tris-HCl
buffer pH 7.7. The homogenates were centrifuged at 600 g for 5 minutes,
then the supernatant fluid was centrifuged at 49,000 g at 4.degree. C. for
15 minutes. The pellets were re-suspended in 5 ml of 0.05 M Tris-HCl
buffer pH 7.7 and incubated at 37.degree. C. for 30 minutes, then
centrifuged at 49,000 g at 4.degree. C. for another 15 minutes. The final
pellet from each rat brain was then re-suspended in 40 ml of 0.05 M
Tris-HCl buffer pH 7.7 containing 100 .mu.g/ml of Bacitracin and used for
the binding assays.
The binding experiments were performed either at 25.degree. C. for 20
minutes or at 0.degree. C. for 3 hours. The reaction mixtures contained 1
ml of the rat membrane preparation as described, 1.5 nM of .sup.3
H-methionine enkephalinamide (38 Ci/m Mole) or .sup.3 H-naloxone (25 Ci/m
Mole) together with bacterial extracts from p.beta.gal or p.beta.gal-end
transformed cultures (4.5 mg protein for enkephalinamide rections and 0.65
.mu.g protein for naloxone reactions). At the end of the binding reaction
the mixture was filtered under vacuum over glass fiber filters (Whatman,
Clifton, N.J., GH-B filters). The filters were washed with a large volume
of cold 0.05 M Tris buffer pH 7.7 and placed in scintillation vials
contining 12 ml of Hydromix (TM Yorktown Research, South Hackensack, N.
J.) scintillation fluid and placed in a scintillation counter to determine
the amount of radioactive agonist or antagonist bound. The results are
shown in the following
TABLE 1
______________________________________
E. coli RR1 Radioligand
.sup.3 H--naloxone
(antagonist)
.sup.3 H--ala-enkephalinamide
%
(agonist) .mu.g inhi-
Plasmid Source .mu.g protein
% inhibition
protein
bition
______________________________________
p.beta.gal-end
pellet 5, 10 84, 100 0.7 68
p.beta.gal
pellet 4 65 0.6 25
______________________________________
It can be seen that .beta.-endorphin from the cell pellet fraction of
bacteria transformed with p.beta. gal-end is an effective inhibitor of th
binding of both an agonist and an antagonist to opiate receptors of the
brain. The displacement of both the opiate agonist enkephalinamide and the
opiate antagonist naloxone was greater in material obtained from the cell
pellet fraction after sonication than in the total bacterial cell extract.
EXAMPLE 7
The Effects of .beta.-Endorphin on Prostaglandin E.sub.1 -Stimulated Adenyl
Cyclase Activity in Neuroblastoma-Glioma Hybrid Cells
The neuroblastoma-glioma hybrid cell line NG 108-15 is richly endowed with
opiate receptors and possesses an adenylate cyclase which can be
stimulated by prostaglandin E.sub.1 (Klee, W. and Nivenberg, M., Proc.
Natl. Acad. Sci., 71, 3474 (1974); Hamprecht, B. and Schultz, J.
Happe-Selyers Z. Physical Chem., 354, 1633 (1973). This stimulation is
inhibited by morphine and the inhibitory affect of morphine can be
prevented by naloxone, a morphine antagonist (Trober, J. et al., Nature,
253, 120 (1975). The .beta.-endorphin preparation used in these
experiments was obtained from fully-induced cells transformed either with
p.beta.gal or p.beta.gal-end, using the pellet fraction obtained after
sonication, as described in Example 3, without further purification by
adsorption to glass. The neuroblastoma-glioma hybrid cells were grown in
T-flasks (1 flask for 60 assays) in Dulbecco's modified Eagle's medium
(Dulbecco, R., Virology 8, 369 (1959)) containing 10% (v/v) fetal calf
serum, 0.1 mM hypoxanthine, 10 .mu.M aminopterin and 16 .mu.M thymidine.
At confluency, cells were harvested and homogenized in 0.32 M sucrose, 40
mM HEPES and 2 mM EDTA, pH 7.6.
The test reaction measured the amount of cyclic AMP synthesized by the
cultured cells in the presence of prostaglandin E.sub.1 measured in the
presence and absence of .beta.-endorphin prepared according to the
invention as described in Example 3 (without purification by glass
extraction). The base line level of adenylate cyclase stimulated by
prostaglandin E.sub.1 was determined in the absence of any opiate agonist
or antagonist. Inhibition of the stimulated adenylate cyclase level was
observed when the incubation was carried out in the presence of a known
opiate agonist or in the presence of an extract of cell sonicate pellet
material prepared from fully induced E. coli RR1 bearing the
p.beta.gal-end plasmid and treated as described above to release the
.beta.-endorphin. Further, the ability of naloxone, an opiate antagonist,
to reverse the effect of .beta.-endorphin was tested. As a control, the
test was run using a similarly-treated sonicate pellet of fully induced E.
coli RR1 bearing p.beta.gal. The incubations were carried out for 15
minutes at 30.degree. C. in a total reaction volume of 100 .mu.l
containing .sup.32 P-ATP, 3.times.10.sup.6 cpm, 10 units creatine
phosphokinase, 50 .mu.M prostaglandin E, 5 .mu.l of opiate agonist or
pellet extract (3.5 .mu.g protein) as described in Example 3, 20 mM HEPES,
5 mM MgCl.sub.2, 1 mM AMP, 20 mM creatine phosphate, 0.1 mM ATP, 0.125 mM
ZK 62711, a phosphodiesterase inhibitor (Schilling, West Germany), and 1
mM of protease inhibitor (Sigma Chem. Co.). At the end of the incubation
period, the reaction was stopped by the addition of 150 .mu.l of 1 N
HClO.sub.4, following which 0.3 ml of water containing 30,000 cpm of
tritiated cyclic AMP was added per tube as a marker. The tube contents
were mixed and centrifuged, following which the supernatant solution was
fractionated by column chromatography by Dowex 50 (TM Dow Chemical
Company, Midland, Mich.) and alumina to recover .sup.32 p-cAMP synthesized
in the reaction (White, A. A., and Kan, D. B. Anal. Biochem, 85, 451
(1978). The amount of cAMP synthesized, from which the adenylate cyclase
activity was inferred, was measured by quantitative determination of the
.sup.32 P counts. The results, expressed as percent inhibition of the
prostaglandin E.sub.1 stimulated activity measured in the absence of added
opiate agonist, are shown in the following
TABLE 2
______________________________________
Inhibition (%)
Plasmid .mu.g Protein
- Naloxone + Naloxone
______________________________________
p.beta.gal-end
3.5 23 0
p.beta.gal
3.0 5 0
______________________________________
.beta.-endorphin, prepared from the cell sonicate pellet of bacteria
transformed by p.beta.gal-end inhibited the adenylate cyclase activity,
and the inhibition was reversed by the addition of naloxone. By contrast,
the cell pellet fraction derived from bacteria transformed by p.beta.gal
did not significantly inhibit the base line adenylate cyclase activity.
EXAMPLE 8
Synthesis of Human .beta.-Endorphin
The principles of the present invention are applied in combination with
prior art techniques to provide a modified coding sequence whose
expression yields human .beta.-endorphin. The method takes advantage of
the fact that the coding sequence for mouse .beta.-endorphin contains a
HhaI site (GCGC) spanning amino acids 26 and 27 of the mouse
.beta.-endorphin sequence. A sequence coding for human .beta.-endorphin
may be constructed by cleaving the mouse sequence with HhaI endonuclease,
followed by ligation of the chemically synthesized nucleotide sequence
coding for the 5 C-terminal amino acids of human .beta.-endorphin.
The mouse coding sequence, prepared as described in Example 1 through the
S.sub.1 nuclease digestion step, is treated with 5 units of HhaI
endonuclease for 1 hour at 37.degree. C. in the manufacturer's recommended
reaction buffer. The DNA is separated from a reaction mixture by phenol
extraction and ethanol precipitation, as described in Example 1. The DNA
is then joined to a synthetic oligonucleotide having the structure
##STR2##
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