Note: Descriptions are shown in the official language in which they were submitted.
-2-
This invention relates to recombinant DNA
techniques.
More particularly, in its broadest aspect,
the present invention relates to a process for the
preparation of oligopeptidPs or polypeptides by
recombinant DNA techniques using a synthetic gene
coding for a repeating polymer of the desired
oligopeptide or polypeptide.
The present invention also relates to such a
synthetic gene and to the preparation thereof.
The synthesis of peptides by chemical means is
a laborious process, which becomes progressively less
efficient with increasing length of the peptide. On
the other hand t the preparation of peptides by micro-
organisms in a fermentation process does not vary inefficiency in relation to the length of the peptide
product. However, in the organisms used in recombinant
DNA processes, short peptides are metabolically unstable.
It is known, for example, that ~he peptide somatostatin,
which is composed of 14 amino acids t iS not stable
when produced directly in E. coli. Stability is
achieved in that case by linking the peptide to a
normal E. coli. protein and subsequently cleaving this
fusion product. (Itakura, K., et al, Science, 198,
1056 - 1063, (1977)).
In the present process, stability and increased
yield are achieved by the use of a synthetic gene
containing multiple repeats of the sequence coding
for a desired small peptide. The product is thus
a large molecule which is metabolically more stable
in E. CQli than the small peptide. Subsequent cleavage
of the large molecule gives the smaller peptide
contained within it and in higher molar yield than
would be obkained if the small peptide had been
prepared directly using a monomeric gene even if the
small peptide were metabolically stable.
In one embodiment, the present invention relates
to a synthetic gene which comprises, in the coding
strand, codons for the amino acids of a desired peptide
in tandem repeat.
By the term "tandem repeat" in this context
is meant the repeat of the sequence of codons in a
linear progression along the DNA molecule with the same
polarity and without interruption.
The complementary or non-coding strand of the
synthetic gene contains a sequence specified by the
sequence of the coding strand according to the accepted
rules of nucleic acid base pairing.
In another embodiment, the present invention
relates to a process for the preparation of such a
synthetic gene which compxises the self assembly
and ligation and optionally repair of a plurality of
appropriate oligonucleotides, the assembly to give the
repeating character being achieved by self-complementary
--4--
overlapping ends at the termini of the group of
oligonucleotides which, when assembled, comprises
the repeating unit of the gene.
In a further embodiment, the present invention
relates to a plasmid vector which comprises inse~-ted
therein at an insertion site adjacent to a bacterial
promoter and downstream from a prokaryotic ribosome
binding site such a synthetic gene such that the gene
is under bacterial promoter control.
In yet another embodiment, the present invention
relates to a cell which has been transformed by having
inserted therein such a plasmid vector.
In a further embodiment, the present invention
relates to a process for the expression of a peptide
which comprises culturing such a cell.
As indicated above, the strands of the synthetic
gene are prepared from a series of short oligonucleotides
which themselves are prepared by joining together the
required nucleotides by known methods in an order
specified by the codons of the amino acids of the
desired peptide and the rules set out in more detail
below.
The oligonucleotides of the coding strand and
the complementary strand of the gene form an over-
lapping set corresponding to the following general
structure:-
~9~
.,
l ~1 cl dl e f 1 3
_ I
3 ,~ r~ -- 5
2 1 2 i 2 1 2 2 12
b c d e f a
The structure shown in this illustration is
composed of six oligonucleotides, three in each strand.
The structure may, however, be assembled from any
number of pairs of oligonucleotides from one pair
upwards, provided:-
(l) that the paired regions designated al
and a~, bl and b2, cl and c2 .,, contain
nucleotide base sequences which are comple-
mentary within the pair, but not to the
sequencesof any other pair;
~2) that each of these regions al, a2, bl, b2,
cl, c2 ,., contain at least four nucleotide
bases;
and
(3) that the sum of the nucleotide bases in
each strandl which when assembled eventually
comprises the repeat length of the polymer,
is divisible by three.
When a set of oligonucleotides of this character
are mixed, the complementary regions of the oligonucleotide
pairs hybridize ~o form structures of high mo].ecular weight
in which the sequences of the oligonucleotides (al-fl in
the above illustration) are repeated in tandem from a few
times to many times, in different molecules. In the
subsequent process, it is desirable to maximise the
number of polymers which have in the sequence al at
the 5' termini thereof. This is achieved by mixincJ
the oligonucleotides al bl cl dl el fl b2 c2 and
d2 e2 of the above illustration in equimolar amounts
and subsequently adding the oligonucleotide f2 a2 in
a less than equimolar amount. Since the complements of
al and fl are present in sub-molar amounts, most
polymers will terminate in al and fl. In the general
case, the 5' terminal oligonucleotide of the comple-
mentary set is adaed to the polymerization mixture
in an amount which is less than equimolar with respect
to the other oligonucleotides, all of which are present
in equimolar amounts.
The "nicks" in the chains, where adjacent
oligonucleotides in the chain juxtapose, may be joined
by the use of the enzyme DNA ligase (EC 6.5.1.1), a
known procedure.
The thus-prepared molecules will have single
stranded ends. These may be converted to the so-called
"blunt end" form by use of the enzyme DNA polymerase
I (EC 2.7.7.7) by a known procedure.
The resultant molecules may be cloned in competent
microbiol cells, in particular E coli, using a desired
vector. In particular, a plasmid vector selected from
1 the so-called "pWT series", see, for example, Tacon,
et al, ~olec. Gen. Genet., 177, 42 " (1980), may be used,
selection of the vector being made such that the synthetic
gene, when inserted in the correct orientation, is read in
the desired transl~tion reading frame.
ReEerence is now made to the following description
and the accompanying drawings in which:
Figure 1 represents a scheme for the production of
the recombinant plasmid pWT 121 containing the repeating
gene for (asp-phe) in the correct DNA-triplet-codon phasing
for expression of the (asp-phe) protein.
Figure 2 is a diagrammatic representation of plas-
mid p~T 121 containing restriction sites, the tetracycline
resistance gene, ampicillin resistance gene and portions of
the tryptophane operon.
Figure 3 represents a scheme for the synthetis of
the dodecanucleotides using tries-ter methodology.
Figure 4 represents an autoradiograph of an SDS-
polyacrylamide gel electrophoresis containing 14C-labelled
proteins synthesized in an E. coli carrying the pWT 121 plasmid.
Figure 5 represents an autoradiograph of a silica
thin-layer chromotography plate containing separated products
of a subtilisin-digestion of the 14C-labelled (asp-phe)
protein.
Figure 1 of the accompanying drawings illustrates
a scheme for the produckion of a recombinant plasmid (pWT
121 (asp-phe)n) which contains the repeating gene for
. ~, ~ ) .
, ~
9~
1 (asp-phe~n in -the correct DNA phasing for expression of an
(asp-phe)n protein~ The plasmid DNA, shown at the top of
the diagram, is cut with the restriction enzyme Hin d III
and then repaired wlth E. coli DNA polymerase I to produce
a blunt ended molecule. The polymer coding :Eor (asp-phe)n,
shown at the bottom of the diagram, has its initially-
protruding 5' -end repaired similarly to produce another
blunt ended molecule. The joining of these two blun-t ended
species with T4 DNA ligase produces the recombinant DNA,
shown in the centre of the diagram, having the correct DNA
phasing for expression of (asp-phe) .
The cloning of this gene, the selection of
bacterial colonies, the detection of the polypeptide product
of the expression of -the gene and the extraction and puri-
i. L5 fication of the polypeptide may all be achieved by known
procedures.
This polypeptide may then be cleaved either
enzymically using speciEic enzymes, or chemically, for example
by the use of cyanogen bromide to cleave polypeptide chains
at methionine residues.
As will be appreciated by those skilled in the
art, the present invention finds numerous applications. Inter-
alia, the present invention may be applied to the peptide
hormones oxytocin, vasopressin and somatostatin, the enkeph-
alins (opiate pentapeptides) and the appetite-con-trolling
peptides.
For purposes of illus-tration one particular embodi-
ment of the present invention will now be described in more-
detail with reference to the sweetner "Aspartame".
- 7a -
.
1 Aspartame is a low calorie artificial sweetner
~ which may be described chemically as aspartyl-phenylalanine~
,4
- 7b -
.~ . . ~
methyl ester. Aspartame is currently produced by a
multi-stage chemical synthes.is.
In view of its use as a sweetener, the total
consumption of Aspartame i5 potentially very large and
there :is, therefore, considerable interest in developing
preparations whereby the material may be obtained
conveniently and on a large scale.
It has been found that Aspartame may be prepared
by a potentially large scale process based upon
recombinant DNA techniques whereby a synthetic gene
coding for (aspartyl-phenylalanine)n is inserted into a
cloning vector having a controllable bacterial promoter
upstream of and substantially adjacent to the insertion
site and the vector may be used to transform bacterial
cells from which the expressed polypeptide may be obtained
and this may then be cleaved to obtain aspartyl-
phenylalanine and hence AspartameO
Accordingly, in a further embodiment, the present
invention relates to a synthetic gene for the expression
of the repeating polypeptide (aspartyl-phenylalanine)n
which comprises a double-stranded DNA molecule comprising
in the coding strand at least two nucleotide trimers
coding for the expression of aspartic acid ~Asp) and,
alternating therewith, at least t~o nucleotide trimers
coding for the expression of phenylalanine (Phe) and
in the other strand repeating and alternating nucleotide
sequences complementary to the (Phe) and (Asp) coding
sequences.
- ~9 -
The coding strand of the synthetic gene, i.e.
that coding for the repeating polypeptide (Asp-Phe)n,
may therefore by represented as coding:
5' - (Asp - Phe - Asp - Phe)n ~ 3'
S where.in n represents an integer, e.g. of up to several
hundred.
There lare two possible codons for Phe, viz
(T~T-T) and (T-T-C), and two possible codons for Asp,
viz (G-A-T) and (G-A-C), and these may be used in any
permutation with corresponding complementary sequences
in the other strand. For example, the coding strand
may be a nucleotide sequence as follows:
Phe Asp Phe Asp Phe Asp
5' -T-T-T-C-G-A-C-T-T-C-G-A-T-T-T-C-G-A-C-T-T- 3'
with a complementary strand having the sequence
3' -A-A~A-G-C-T-G-A-A-G-C-T-A-A-A-G-C-T-G-A-A- 5'
In this case, (T-T-C) is used as the sequence coding for
phenylalanine and both of the codons (G-A-C) and (G-A-T)
alternate for aspartic acid.
The other possible permutations and combinations
of nucleotide se~uence for the coding and complementary
strands will be clear to those skilled in the art.
The synthetic "Aspartame gene" according to
the present invention maybe prepared by the polymer-
isation of a double stranded fragment of DNA consisting
of two dodecanucleotide strands, one having the sequence
for expression of (Asp-Phe)2, the coding strand and the
-10~ 6~
other being complementary to it, the complemen-tary
strand.
Accordingly, in a further embodiment, the present
invention relates to such a process which comprises
providing a first dodecanucleotide sequence coding for
aspartyl-phenylalanine by linking together appropriate
nucleotide monomers, providing a second dodecanucleotide
sequence complementary to the first sequence, phosphoryl-
ating both dodecanucleotides using T4 polynucleotide
kinase and mixing the first and second sequences together
to form a double-stranded DNA structure.
The dodecanucleotide strands may each be
synthesised from the respective monomers which are
protected by blocking groups in known manner and linked
by conventional phosphotriester methodology, (see, for
example, Hsiungl et al, Nucleic Acids Research, 6, 1371 -
1385, (1973)), the dodecamers so obtained are deblocked
and subsequently purified. Following purification and
phosphorylation with T4 polynucleotide kinase (E.C. 2.
7.1.78), the coding and complementary strands are mixed
together with the result that double stranded structures
are fo~led by hydrogen bonding. It has been found that,
by using the dodecamer coding for (Asp-Phe)2 for the
coding strand and a corresponding dodecanucleotide
complementary strand, very stable double-stranded
structures are obtained resulting from the six base
overlap of the strands, which, for the example
previously given, is represented as follows:
S' pT-T-T-C-G-A-C-T-T-C-G-A 3'
3' G-A-A-G-C-T-A-A-A-G-C-Tp 5'
Linear polymerisation of the above double-stranded
structures is achieved by incubation with T4-DNA
ligase (E~ 6.5.1O1) to yield long polymers of random
length. After separation of unligated material, it is
found that a range of po]ymers is obtai.ned containing
from 2 to 500 and more repeats o~ the basic units.
The present invention also relates to a process
for the expression of taspartyl-phenylalanine)n which
comprises inserting such a synthetic gene into an
insertion site of a plasmid vector reading in the
correct phase for the inserted gene, the insertion
site being adjacent to a bacterial promoter and
downstream from a prokaryotic ribosome binding site,
transforming competent microbial cells using the
resulting plasmid vector, culturing the transformed
cells and harvesting the expressed peptide. Moreover, the
polymeric product is considered novel.
The present invention further relates to a
process for the preparation of Aspartame (the methyl
ester of aspartyl-phenylalanine) which comprises
inserting the above-mentioned synthetic gene into a
plasmid cloning vector, e.g. pWT 121, designed to read
in the correct phase for expression of the inserted
gene and having a bacterial promoter upstream of and
6~
-~2
adjacent to the insertion site. The plasmid vector
may be used to transform E~ coli HB 101 cells, the
expressed ~Asp-Phe)n harvested and suhjected to
enzymatic cleavage using an enzyme specific for amino
acids with aromatic side chains, such as subtilisin
(E.C. 3.4.4.16), chymotrypsin (EoC~ 3-4-4-5! or
proteinase K (E.C. 3.4.21.1~), to obtain aspartyl-
phenylalanine which is methylated to produce Aspartame.
The enzyme used may be in a soluble form or, preferably,
immobilised on a solid support. Methylation may either
be carried out by conventional chemical means or by an
exchange reaction with the enzyme in the presence of
methanol or by direct enzymatic methano]ysis of the
polymer.
It will be appreciated that, given the triplet
nature of the genetic code, the gene may be read in any
one of three phases and it is therefore essential for a
gene to use as the plasmid vector one providing for
translation in the correct reading frame.
It has been found that a preferred plasmid
vector for use in the present process is one selected
from the so-called "pWT series" mentioned above.
Basically, the pWT series plasmids comprise a
Hin d III (E.C. 3.1.23.21) restriction site adjacent
to a cloned E. coli tryptophan promoter. By Hin d III
restriction and the insertion of Hin d III linkers it is
possible in the pWT series to construct plasmids able
-13-
to translate in all thr~e reading frames these being
pWT 111, pWT 121 and pWT 131 as disclosed in the above-
mentioned reference.
Transcription and -translation of inserted DNA
in the pWT series plasmids is under direct and strong
tryptophan control; thus, in the presence of tryptophan,
the operon is repressed, while, in the absence of
tryptophan, it is de-repressed.
Plasmids of the pWT series also possess, in
the region immediately following the Hin d III site,
the gene for tetracycline resistance so that, after
insertion of the DNA, transcri.ption and translation may
easily be confirmed since, when it has occurred,
tetracycline resistance is maintained.
In a preferred embodiment of the present process,
therefore, the synthetic gene is inserted into the
appropriate plasmid of the pWT series, that is the
plasmid designated "pWT 121". This plasmid is illustrated
diagrammatically in Figure 2 of the accompanying drawings
and has the following characteristics:
A molecular length of 4837 bp; a Hpa I (E.C.
3.1.23.23)site; a Hin d III site 206 bp from the ~
site; a Bam HI (E.C. 3.1.23.6) site 353 bp from the
Hin d III site; a Sal I (E.C. 3.1.23~7~ site 275 bp from
the Bam HI site; a Pst I (E.C. 3.1.23.31) site 2958 bp
from the Sal I site and 1045 bp from the ~ I site; the
gene for tetracycline resistance extendiny from the region
-14-
of the Hpa I site to beyond the Sal I site; the gene
for ampicillin resistance in the region of the PstlI
site and the cloned portion of the trp operon comprising
the region between the promoter and the first portion of
the E gene between the Hpa I and the Hin d III si-tes.
The synthetic Aspartame gene according to the
present invention was cloned into pWT 121 as follows:-
The plasmid pWT 121 was restricted with theenzyme Hin _ III to yield a linear molecule which was
then treated with DNA polymerase and all four
deoxyribonucleoside triphosphates to produce completely
base paired 'blunt ends'. The blunt-ended molecule was
then treated with bacterial alkaline phosphatase (E.C.3.1.3.1)
to remove the 5' phosphates. The plasmid is now
prepared for insertion of the synthetic gene which is
first treated as follows:-
The synthetic gene is treated with E. coli DNApolymerase and all four deoxyribonucleoside triphosphates
to produce blunt ends and the "repaired" genes separated
from enzyme and small molecules.
Blunt ended vector DNA and blunt ended gene
material are mixed in a from 1:1 to 1:2 ratio and
ligated together with high concentrations of T4-~NA
ligase to produce the series of plasmids "pWT 121/(asp-
phe)n". The sizes of the cloned inserts,, determinedby restriction enzyme digestion, wexe found to range
from 60 to 900 bp (i.e. from 5 to 75 repeats of the
dedecanucleotide unit).
The pWT 121/(asp-phe)n plasmids prepared as
described above are used to transform E. coli cells
in known manner, for example by exposure of cells
treated with calcium chloride to the pWT 121/(asp-phe)n
p].asmid. TransEormed cells cultured in known manner in
medium containing no tryptophan and preferably
containing the inducer ~-indolearcylic acid, expressed
a protein consisting essentially of (asp-phe)n which,
afcer enzymatic cleavage and methylation, produced the
desired aspartyl-phenylalanine methyl ester, AspartameO
As mentioned above, the product Aspartame is
conventionally obtained by a multi-stage chemical
synthesis. An intermediate used in this conventional
synthesis is L-phenylalanine. The present invention
also relates to the preparation of L-phenylalanine
useful in this conventional synthesis. When produced
by chemical synthetic means, phenylalanine is obtained
as a racemate which needs to be resolved before the
L-isomer may be obtained and this requires an additional
and expensive stage in the process. When obtained by an
embodiment of the present invention, the phenylalanine
is produced directly as the I.-isomer.
Accordingly, the present invention further relates
to a process for the preparation of L-phenylalanine which
comprises treating the (asp-phe)n peptide, isolated
from a culture of E. coli cells carrying plasmids
incorporating the synthetic Aspartame gene, with an
-16~
acidic or enzymatic hydrolysing agent and separating
from the hydrolysate the desired L-phenylalanine.
This process, of course, also gives rise to
L-aspartic acid and the present invention also relates
to such a process.
Preferred hydrolysing agents include hydro-
chloric acid or carboxypeptidase ~E.C. 3.4.12.x),
aminopeptidase (E.C. 3.4.11.x) or non specific endopeptidases.
Separation of the hydrolysis products may be
achieved by known methods.
The following Examples illustrate the present
invention:
EXAMPLE 1 (asp-phe)n
S~nthesis of dodecanucleo~ides
The two dodecanucleotides, TpCpGpApApApTpCpGpApApG
and TpTpTpCpGpApCpTpTpCpGpA, were synthesised by
conventional triester methodology as described, for example,
by Hsiung, Loc cit, and in accordance with the reaction
scheme illustrated in accompanying Figure 3 wherein N
ts GiSobu T CbZ or AbZ. The fully protected
dodecanucleotides were deblocked with 2% w/v benzene
sulphonic acid, 0.1 M tetraethyl ammonium fluoride in
THF/pyridine/water (8:1:1 by volume), followed by
treatment with ammonia. The de-blocked dodecanucleotides
were purified by HPLC on "Partisil 10 SAX", (micro-
particulate silica which has been derivatised with
quaternary ammonium groups (Whatman)).
-17-
~ risation of dodecanucleotides
Each synthetic dodecanucleotide (2 ug) was
phosphorylated in a 10 ,ul reacti.on conta.ining from 10
to 50 ~Ci o~ ~ 3 P-ATP, 50 mM Tris-Cl pH 7~8, 5 mM
magnesiurn chloride, 0.25 mM unlabelled ATP, 10 mM
mercaptoethanol and 3 units (1 unit is the amount that
causes the transfer of 1 nanomole of 32P-phosphate from
~(32P)-ATP to the 5' hydroxy terminus of a polynucleotide
in 30 minutes at 37C under standard assay conditions, see,
for example, Richardson, Nucleic Acids Research, 2, 815)
of T4 polynucleotide ~inase (E.C. 2.7.1.78). After 60
minutes at 37C the two dodecanucleotides were mixed,
unlabelled ATP was added to bring the ATP up to 1 mM
together with 0.1 units (1 unit is the amount that will
convert 100 monomoles of d(A-T)1000 to an exonuclease III-
resistant form in 30 minutes at 30C under standard assay
conditions, see, for example, Modrich and l,ehman, J.
Biol. Chem., 245, 3626 (1970)) of T4 DNA ligase (E.C. 6.5.
1.1) and the reaction mixture incubated for 24 hours at
25C. ATP and unligated nucleotide monomers were
removed by passage down a "Sephadex G-50" (superfine,
particulate cross~linked modified dextran polymer
(Pharmacia)) column (20 cm x 0.8 cm) in 50 mM sodium
chloride, 10 mM Tris-Cl pH 7.5, 0.2~ w/v sodium
dodecylsulphate (SDS). The double-stranded polymers
were concentrated by ethanol precipitation. The product,
after separation, was found to be a mixture of polymers of
6~
-18-
random length containing from 2 to more than 500
repeats of the basic units.
Cloning of the synthetic gene
(a) slunt end repair of the synthetic gene
2 ,ug of polymer prepared as described above
was incubated in a 40 ~1 mixture with 50 mM Tris-Cl
pH 7.8, 5 mM magnesium chloride, 1 mM mercaptoethanol,
0.125 mM of each of the four deoxynucleotide triphosphates
and 2 units (1 unit is the amount that causes the
incorporation of 10 nanomoles of nucleotide into an
acid precipitable form in 30 minutes at 37C under
standard assay conditions using poly d (A-T) as template,
see, for example, Richardsonl et al, J. ~iol. Chem.,
239, 222, (1964)) of E. coli DNA polymerase I (E.C. 2.7.
7.7). The mixture was incubated for 20 minutes at 10C
and the mixture used without further purification.
(b) Preparation of the cloning vector pWT 121
50 ~g of pWT 121 DNA was digested with a two fold
excess of ~in d III (E.C. 3.1.23.21). The mixture was
extracted twice with an equal volume of phenol and
precipitated with ethanol.
Repair with E. coli DNA polymerase to generate
blunt ends was carried out as described in (a) aboveO
The blunt ended DNA was then treated with
bacterial alkaline phosphatase (E.C. 3.1.3.1) to remove
terminal phosphates and to prevent the DNA recircularising
during subsequent ligation. The DNA was incubated for 30
--19--
minutes at 37C in the presence of a two-fold excess of
enzyme in 10 mM Tris-Cl pH 7.5, 0.1~ SDS. The mixture
was thereafter exhaustively extracted with phenol,
washed several times with chloroform and then precipitated
with ethanol.
(c) Blunt end ligation
Blunt ended dodecanucleotide polymer from (a)
above and blunt ended pWT 121 DNA from (b) above were
mixed in a 1:1 proportion, by weight, and ligated at
15C with 0.2 units of T4 DNA ligase in a 20 ~1 mixture
containing 50 mM Tris-Cl pH 7.8, 5 mM magnesium chloride,
1 m~ ATP and 10 mM mercaptoethanol. After 24 hours the
pWT 121/(asp-phe)nrecombinant plasmids were ready for use
to transform E. coli cells.
Transformation and gene expression
E. coli K12 HB 101 cells (genotype gal , lac ,
- r _ _
ara , pro , arg , str , rec A , rk ~ Mk ; ~oyer, H.W. and
Roullard - Dussoix, D., J. Mol. Biol., 41, 459-472) were
transformed by the procedure of Katz et al (1973) J.
Bacteriol, 114, 577-591, and plated on L-agar plates
supplemented with ampicillin (100 ~g/ml).
Recombinant clones were purified by streaking
to obtain single colonies. Clones thus isolated were
examined by colony hybridisation on nitro-cellulose
filters (Grunstein and Hogness (1975) Proc. Nat~ Acad.
Sci. U.S.A. 72, 3961-3965) using a kinase-labelled
20-
synthetic dodecanucleotide as a hybridisation probe.
Single colonies positive by colony hybridisation were
grown up to an A600 oE 0.6 in 25 ml of M9 medium
(Miller, (1972), Experiments in Molecular Genetics, Cold
Spring Harbour Laboratory, New York, page 433) supplemented
with ampicillin (100 ~g/ml) and tryptophan (40 ~g/ml).
A 1 ml sample was taken from this repressed culture and
labelled for 10 minutes with 5 ~Ci 4C-amino acids before
being chased for 10 minutes with 200 ul of 20% w/v
"casamino acids" (Difco). The cells were centrifuged
~"pelleted") (10,000 x g for 10 minutes), washed several
times with phosphate-buffered saline containing 100 ~g/ml
gelatin to remove excess label and the final pellet lysed
in 50 ~1 of a buffer (FSB) containing 10% v/v glycerol,
0.01% w/v bromophenol blue, 5% v/v ~-mercaptoethanol,
3% w/v SDS and 65 mM Tris-Cl pH 8 by heating to 90C
for 2 minutes. The remainder of the 25 ml initial culture
was pelleted (10,000 x g for 10 minutes) and resuspended
in an equal volume of M9 medium supplemented with
ampicillin (100 ,ug/ml) and ~-indoleacrylic acid (5 ~g/ml).
After various times of induction 1 ml samples were
withdrawn and labelled as described above. From 5 to
l0 ~1 aliquots of the final lysates were separated on
acrylamide gels (12.5% polyacrylamide + 0.1% SDS, see,
for example, Laemmli, Nature, 227, 680-685, (1970)).
The gels were dried and autoradiographed to locate
the pSitionsof the labelled proteins. By comparing
the patterns from uninduced and induced cells, proteins
synthesised under the control of the tryptophan promoter
could be identi-Eied.
Figure 4 of the accompanying drawings depicts
S an autoradiograph of a 12.5~ acrylamide: 0.1~ SDS gel
on which are separated 14C-labelled proteins synthesised
in E. coli cells carrying recombinant pWT 121. (asp-phe)n
plasmids. Track 1 showns proteins labelled with 4C~
amino acids in the presence of 40 ~lg/ml of L-tryptophan,
i.e. any genes inserted at the Hind III site will be
repressed and no protein should be produced. Track 2
shows proteins labelled with 4C-amino acids in the
absence of L-tryptophan and in the presence of 5 ~g/ml
of ~-indole acrylic acid, i.e. inserted genes will be
fully induced and should express the protein coded for
by the insert. ~The protein which appears strongly in
track 2 has been shown to be a repeating polymer of
(asp-phe).) Tracks 3 and 4 correspond to tracks 1 and
2, respectively, except that twice the amount of protein
was used. The standard protein molecular weights
shown on the right of the illustration are those of a
standard mixture of protein obtained from the
Radioche~ical Centre, Amersham, ~uckinghamshire,
England.
Isolation of labelled proteins from gels:
particular bands of protein were isolated from acrylamide
gels by separating the protein lysate in a series of
-21a-
parallel tracks. One of these tracks was cut from the
gel with a scapel and stained with Coomassie Brilliant
Blue R (Gurr, Searle Diagnostics) while the rest of the
gel was soaked for 20 minutes in 15% v/v glycerol and
stored at -70C. The stained gel slice was dried and
autoradiographed Eor from 24 to 48 hours to define the
position of the labelled bands. This enabled the
relevant portion of the frozen gel to be excised The
gel slices were broken up by forcing them through a
1 ml disposable syringe with no needle and the labelled
protein eluted with 10 ~ triethylammonium bicarbonate
pH 7.5 for 24 hours. Gel fragments were removed by
centrifugation and the supernatant dialysed versus
10 mM triethylammonium bicarbonate. From 50 to 75
recoveries of labelled protein bands were obtained
in this way.
Enzyme digests: Crude cell lysates or isolated
bands from acrylamide gels were digested either with
proteolytic enzymes in solution at concentrations of
from 1 to 10 mg/ml, or with equivalent amounts of
enzyme i~mobilised on a solid support, for periods up
to 72 hours. 10 mM triethylammonium bicarbonate was
-22-
used for digests in the pH range 7-8, while 10 mM
glycine/sodium hydroxide buffers were used up to pH
10.5.
Thin Layer chromatography: Enzyme digests of
labelled proteins were separated on Merck silica gel
TLC plates with concentration zone (20 x 20 cm), using
either n-butanol:acetic acid:water (8:2:2 by volume)
or n-propanol:conc. (0.880) ammonia (7:3 by volume) as
solvent. After development, the plates were dried and
autoradiographed using Kodak "Kodirex" X-ray film to
detect the labelled peptide products. Digests with
chymotrypsin (E.C. 3.4.4.5) or subtilisin (E.C. 3.4.4.16)
immobilised on a solid support or with subtilisin or
proteinase K (E.C. 3.4.21.14) in soluble form all gave
a mixture of products. Among these in each case was
a compound which co-chromatographed with authentic
aspartyl-phenylalanine. (Figure 5 of the accompanying
drawings depicts an autoradiograph of a silica thin
layer chromatography plate on which are separated the
products of enzymic digestion of (asp-phe)n protein.
14C-labelled (asp-phe)n protein isolated from a 12.5%
acrylamide: 0.1% SDS gel was digested for 16 hours at
37C with subtilisin immobilised on a solid support.
The thin layer chromatography plate was developed
with n-propanol:0.880 ammonia (7:3 v/v). The arrows
denote the positions of markers of auth~ntic (asp-
phe) and (phe-asp).) This compoun~ when recovered
9~
~22a
from the TLC plate and hydrolysed in hydrochloric acid
yielded only aspartic acid and phenylalanine.
Hydrolysis of induced protein to produce
L-aspartic acid and L-~henylalanine
14C-labelled (asp-phe)n protein isolated from
an acrylamide gel, or a crude cell lysate prepared by
lysing induced 14C-labelled E. coli cells carrying
pWT 121/(asp phe)n plasmids in a buffer containing 5%
v/v ~-mercaptoethanol, 3% w/v SDS and 65 mM Tris.-C]
pH 8 was used for hydrolysis. A lysate made from 1 ml
Of an
-23-
induced cell culture, or the isolated induced protein
from a similar volume of culture was sealed in a tube
with 1 ml of 6 M hydrochloric acid and heated to llO~C
for 16 hrs. After this time, the tube was opened and
the contents removed and evaporated to dryness.
Examination of the hydrolysate by thin layer
chromatography showed the presence of 14C-labelled
L-aspartic acid and L-phenylalaninel together with
smaller amounts of other amino acids.
EXAMPLE 2
A polymer (asp-phe)n may be cleaved by means
of an enzyme, such as chymotrypsin or subtilisin, which
cuts at aromatic amino acids. Alternatively, a polymer
~asp-phe-lys lys)n may be produced which is cleavable
by trypsin or by an enzyme of similar specificity or
by a combination of enzymes to give the dipeptide asp-
phe. For example, such cleavage at paired basic amino
acid residues is known to be involved in the process
by which hormone precursors are cleaved to active
species, e.g. the corticotropin-~-lipotropin-MSH-
enkephalin system (Nakanishi, et al, Nature, (1979),
278, 423-427).
A gene for such a repeating tetrapeptide may be
produced by the joining of four chemically synthesised
dodecanucleotides:-
(1) A.A.G.A.T.T.T.C.A.A.A.A
(2) A.G.G.A.C.T.T.T.A.A.G.A
-2~
(3) A.G.T.C.C.T.T,T.T.T.G.A
~ 4) A.A.T.C.T.T.T.C.T.T.A.A
to form a repea-ting structure:-
asp phe lys lys asp phe lys lys asp phe
5' A~A~G~A~ToT~T~C~A~A~A~A~A~G~G~A~C~T~T~T~A~A~G~A~A~A~G~A~T~T~T~T~
(2) (1)
' EcoRI'
3' T T C T A A.A.G.T.T.T.T.T.C.C.T.G.A.AA~.T.T.C.T.T.T.C.T.A.A.A.A.5'
(4) (3) (4~ (3)
This entails the use of two codons for each of the amino
acids: G.A.T and G.A.C for asp; T.T.T and T.T.C for
phe: and A.A.A and A.A.G for lys. These are all
acceptable in E. coli. The only restriction enzyme
recognition site within this polymeric gene in that for
EcoRI' (A.G.A T,T.T).
Repair of the above polymeric gene using E. coli
DNA polymerase I gives a molecule which reads in the
correct reading frame on blunt end ligation into the Hin
d III site of the plasmid pWT 111, for example.
The experimental procedure is as described in
Example 1. The required polypeptide obtained on induction
may be cleaved using trypsin, for example, and the asp-
phe dipeptide isolated.
EXAMPLE 3
Sma].l quantities of the tripeptide Pyro-glu-his-
gly have the ability to elicit an anorexic response in
animals, i.e. it causes a reduction in food intake, and
~ ~g~6
-25-
is therefore of interest in the control of appetite
in humans. (O. Trygstad, et al, Acta Endocrinol, 89,
196-208, 1978).
A polymer of the form (glu-his-gly-lys-lys)
would be cleaved using trypsin, Eor example, to yield
the tripeptide glu-his-glyO Glutamic acid may be
converted to pyroglutamic acid by the action of heat,
see, for example, U.S. Patent No. 2,528,267.
Such a repeating polymer is coded for by a
repeating gene composed of four chemically synthesised
oligonucleotides, two tetradecanucleotides and two
hexadecanucleotides:-
(1) A.G.GoA.A.C.A.C.G.G.T.A.A.G.
(2) A.A.A.G.A.G.C.A.T.G.G.C.A.A.A.A
(3) C.T.C.T.T.T.C.T.T.A.C.C.G.T.
(4) G.T.T.C.C.T.T.T.T.T.G.C.C.A.T.G
These oligonucleotides may be phosphorylated and joined by
ligation, using the techniques described above, to give a
structure:-
glu his gly lys lys glu his gly lys
5' A.G.G.A.A.C.A.C.G.G.T.A.A.G.A.A.A.G.A.G.C.A.T.G.G.C.A.A.A.-
_ _
(1) (2)
3' T.C.C.T.T.G.T.G.C.C.A.T.T.C.T.T.T.C.T.C.G.T.A.C.C.G.T.T.T.-
(4) (3) (4)
lys glu his
A.A.G.G.A.A.C.A.C. 3'
-
(1)
T.T.C.C.T.T.G.T G. 5'
(3)
-
-26-
Two codons are used for each amino acid: G.A.A
and G.A.G for glutamic acid; C.A.C and C.A.T for histidine;
G.G.T and G.G.C for glycine; and A.A.A and A.A.G for
lysine. All of these codons are acceptab~e in E. coli.
There are no restriction enzyme recognitiorl sites in
this gene. The experimental procedure is as described
in Example 1. This gene reads in the correct reading
frame when blunt end ]igated into the Hin d III site of
the plasmid pWT 111, for example.
EXAMPLE 4
.
The pentapeptide arg-lys-asp-val-tyr is an
analogue of the hormone thymopoietin, which has activity
in inducing the differentiation of pro-thymocytes to
thymocytes, (Goldstein, et al, Science, (1979), 204,
1309-1310). It has pharmaceutical applicat}on in the
treatment of thymic disorders.
A repeating gene coding for a polymer of this
thymic hormone analogue may be constructed from four
chemically synthesised oligonucleotides, two tetra~
decanucleotides and two hexadecanucleotides:-
(1) A~c~c~G.T.A.A.A.G.A.T.G.T.T.T.A
(2) C.C.G.A.A.A.G.G.A.T.G.T.C.T
(3) T.T.T.C.G.G.T.A.A.A.C.A.T.C.T.T.
(~) T.A.C.G.G.T.A.G.A.C.A.T.C.C
These oligonucleotides may be phosphorylated and
joined by ligation to give a structure:-
-27-
arg lys asp val tyr arg lys asp val
5' A.C.C.G.T.A.A.A.G.A.T.G.T.T.T.A.C.C.G.A.A.A.G.G.A.T.G.T.C.T.-
(1) (2) ~ Acc I
3' T.G.G.C.A.T.T.T.C.T.A.C.A.A.A.T.G.G.C.T.T.T.C.C.T.A.C.A.G.A.-
(3) (4)
tyr
A.C.C.G.T.A. 3'
- ~ (1)
T.G.G.C.A.T. 5'
In this structure single codons are used for
tyrosine (T.A.C) and aspartic acid (G.A.T), while two
codons are used Eor arginine (C.G.T and C.G.A), lysine
(A.A.A and A.A.G) and valine (G.T.T and G.T.C). There
is a single restriction enzyme recognition site within
the gene for the enzyme Acc I. (G.T.C.T.A.C) this site
repeats every 30 base pairs. This gene is designed to
be read in the correct reading frame when inserted by
blunt end ligation into the Hin d III site of -the plasmid
pWT 111, ~or example. The experimental procedure is as
EXAMPLE 5
The enkephalins are naturally occurring peptides,
found in the human brain, which are said to have a role
as pain-killers. They are therefore of considerable
pharmaceutical interest. There are two compounds known,
met-enkephalin, which has the sequence (try-gly-gly-
phe-met) and leu-enkephalin, wherein the terminal
4~6
~28~
methionine is replaced by leucine. This example relates
to met enkephalin, but a similar approach is applicable to
leu-enkephalin. A polymer of (tyr-gly-gly-phe-met-lys-lys)n
may be cle,~ved by trypsin, for example, to give the
required pentapeptide. Such ~ polymer is speciFied by a
repeating cJene constructed from six chemically synthesised
o]igonucleotides, two octadecanucleotides and four
dodecanucleotides:-
(1) G~T~A~T~G~G~ToG~G~A~T~T~T~A~T~G~A~A~
(2) G.A.A.A.T.A.C.G.G.A.G.G.
(3) C.T.T.T.A.T.G.A.A.A.A.A~
(fi) T.A.T.T.T.C.T.T.C.A.T.A.A.A.T.C.C.A.
(5) A.T.A.A.A.G.C.C.T.C.C.G.
(6) C.C.A.T.A.C.T.T.T.T.T.C.
These oligonucleotides may be phosphorylated and
joi.ned by ligation in one of two ways. Either all six
oligonucleotides may be mixed and ligated to give the
required polymer in one step. Alternatively, oiigonucleotides.
1, 2 and 4 and oligonucleotides 3, 5 and 6 may be ligated
separately to give hlocks which may then be mixed and liaated
to produce the same polymer, having the structure:-
tyr gly gly phe met lys lys tyr gly gly
5' G.T.A.T.G.G.T.G.G.A.T.T.T.A.T.G.A.A.G.A.A.A.T.A.C.G.G.A.G.G.C.
(1) Mbo II~ (2) ~nl I
EcoRI'
3' C.A.T.A.C.C.A.C.C.T.A.A.A.T.A.C.T.T.C.T.T.T.A.T.G.C.C.T.C.C.G.
r,\
(6) (4) (5)
29-
phe met lys
T.T.T.A.T.G.A.A.A.A.A. 3'
(3)
A.A.A.T.A.C.T.T.T.T.T.C.A.T.A.C.C. 5'
(6)
In this structure, A.T.G is used to code for
methionine and T.T.T to code for phenylalanine, multiple
codons are used for the other amino acids: tyrosine
(T.A.T and T.A.C); ylycine (G.G.T, G.G.A and G.G.C) and
lysine (A.A.A and A.A.G). There are single reco~nition
sites within the gene for the enzymes Mnl I (C.C.T.C),
Mbo II (G.A.A.G.A) and EcoRI' (G.G.A.T.T.T) as shown
above. These sites repeat every 42 bases. The gene is
designed to be read in the correct reading frame when
inserted by blunt end ligation into the Hin d III site of
the plasmid pWT 121, for example.
