Note: Descriptions are shown in the official language in which they were submitted.
06208
Background to the Invention
This invention is concerned with enhancing the
effective expression and stabilizing desirable polypep-
tides when they are produced in microorganisms.
Low molecular weight proteins such as insulin,
proinsulin produced in E. coli using recombinant DNA have
been found to be rapidly degraded unless these proteins
were fused to a large E. coli protein. The most commonly
used is the lac system which consists of a 1007 amino
acid truncated ~-galactosidase for the production of
large amounts of a desired protein. Previously we found
that when proinsulin was fused with a portion of the
coding sequence for ~-galactosidase which codes for 450
or 590 amino acids, 30% of the E. coli protein was
represented by the fused product. A major drawback of
this approach is that the desired product constitutes ~
only a small portion of the hybrid polypeptide resulting
in reduced yield and increased difficulty in purification.
Efforts to reduce the size of leader sequence usually
renders the product unstable. Another strategy followed
by Garvin et al in Canadian Patent Application 563,270
of 1 May 1984 to prevent degradation of human proinsulin
in E. coli is by expressing the multimeric proinsulin
coding sequence to produce a stable multidomain poly-
peptide which can be converted to a monomeric proinsulin
,
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13~6~08
unit by cyanogen bromide cleavage. The disadvantage
is the formation of a proinsulin analog containing 4
extra amino acids which need further protein processing.
We have found that these disadvantages may
be overcome with our novel finding that a DNA sequence
comprising (a) a sequence coding for a desired poly-
peptide, for example insulin, proinsulin, especially
human proinsulin, somatostatin or one of the interferons
and; (b) a selected leader signal sequence coding
for a range of from a tri-peptide to about one third o~ the
size of peptide coded by sequence (a), said selectcd
leader signal sequence being chosen to enhance the
effective expression of, and/or stabilize the polypeptide
coded by sequences ~a) an~ (b) when said DNA sequence
resides and is expressed in a foreign host microorganism.
Sequence ~b) preferably codes for a homo-
amino acid sequence and also preferably codes for 5-10
amino acids. We have found that if sequence (b) codes for: 7
amino acids it is especially useful. Particular examples
of the DNA sequence comprise sequence (a) coding for
human proinsulin and sequence (b) coding for a homo-
amino acid sequence 7 residues long, the amino acid
being selected from glutamine, cysteine and serine.
Such DNA sequences may be incorporated in a cloning
vehicle and a particular DNA-cloning vehicle combination
.
1306~Q~
found useful is a plasmid of the pNS:LY7 type. These
DNA sequences may be incorporated into microorganism,
for example, E. coli. In some instances the polypeptide
product of sequence (b) of the DNA sequence may have
the additional property of being recognised by an affinity
column medium.
A method of making a modified microorganism
capable of producing an increased yield of a desired
foreign polypeptide may comprise:
i) ligating a DNA sequence coding for the desired
polypeptide ~equence (a)) to an opened cloning vehicle
sequence;
ii) ligating, to the DNA sequence resulting from
step i) upstream of sequence (a), a selected leader
signal sequence (sequence (b)) coding for a range of
from a tri-peptide to about one third of the size of
peptide coded by sequence (a) and;
iii) placing the cloning vehicle resulting from
step ii) in a suitable host microorganism and recovering
the modified microorganism. A particular example employs
E. coli (JM 103), sequence (a) codes for human proinsulin
and sequence (b) codes for a homo-amino acid sequence 7
residues long, the amino acid being selected from
glutamine, cysteine and serine.
A biological method of producing a desired
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polypeptide comprises:
iv) culturing the modified microorganism
resulting from step iii) above in conditions that allow
for the repression of the gene coding for the desired
polypeptide;
v) isolating the polypeptide coded by sequences
(a) and ~b);
vi) cleaving the polypeptide resulting from step
v) to yield a polypeptide coded by sequence (a) and a
polypeptide coded by sequence (b), and;
vii) purifyin~ the product of step (vi) to yield
the desired sequence (a) polypeptide in amounts increased
over those obtained in the absence of the leader signal
sequence.
A particular example of this biological
method of producing a desired polypeptide concerns the~
production of human proinsulin using E. coli (JM 103),
sequence (b) codes for a homo-amino acid sequence 7
residues long, the amino acid being selected from
glutamine, cysteine and serine, and step vi) comprises
cyanogen bromide cleavage.
Description of the Drawings
Figure 1 shows electrophoretic migration
patterns when compared with Marker IM) proteins (lane 1)
The products of E. coli strains with different leaders
to the proinsulin gene are shown in lanes 2, 3, 4, 5 and
can be compared with the E. coli (JM 103) control in
lane 6.
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Example
Leader fragments 2S mer were synthesized by both
the solution-phase phosphotriester method and solid-phase
phosphoamidit~ method with an Applied Biosystem Synthesizer
followed by purification with 12% polyacrylamine gel with
7M urea. Temperatures are in C.
Cloning of proinsulin DNA: Construction of pNS:LY7
Plasmid pSI-BCA4 (40 ~g) was digested for 2 hr at
37 with 40 units of Eco RI and 40 units of Bam HI. The
proinsulin gene was isolated by polyacrylamide gel.
Approximately 200 ng of the proinsulin gene was ligated
with 1 ~g of pUC8 plasmid DNA previously cut with Eco RI and
Bam HI. Transfectants were recognized by the loss of ~-gal-
actosidase activity. An example of this procedure is shown
on page 6. A shows the schematic procedure and B shows the
resulting code sequences around the leader sequences, co~ding
in this instance for serine and leucine. Plasmids of trans-
fectants were obtained by the mini preparation procedure
(Bethesda Research Laboratory (BRL)), and the DNA sequence
was determined by the dideoxy method.
Insertion of synthetic leaders 25 mer at Eco RI site of vector
pNS:LY7: Construction of Plasmids pNS:LY7 (qln or cys)
6~c~ibmoTtrr~ leader oligonucleotides (25 mer, 0.3
Con1p~ e~
pmole, 1 ~ ere phosp~orylated separately in 2.5 ~1 of H2O,
0.5 ~1 of lOX kinase buffer, 0.5 ~1 of T4 kinase and 0.5 ~1
of 1 mM ATP at 37 for 45 min. Then the kinased solution of
the complimentary oligonucleotide was combined and heated at
70 for 10 min before it was cooled to room temperature slow-
ly. Then 50 ng (1 ~1) of plasmid pNB:LY7 previously cut by
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Eco RI and dephosphorylated with calf intestinal
alkaline phosphorylase, 1 ~1 10X kinase buffer, 1 ~1
li~ase and 1 ~1 4 mM ATP were added and were incubated
at 12 for 18 hr. An aliquot (11 ~1) of the ligated
solution was transformed with 200 ~1 of competent JM
103 at 0 for 30 min, followed by heat shock of 2 min
at 42. The 2YT medium (800 ~1) was added. The culture
medium was incubated at 37 for 1 hr. An aliquot
(100 ~1) was plated on Yeast-Tryptone (YT) plate with
ampicillin and incubated at 37 for 18 hr. Generally
thirty colonies were observed compared to one in the
control experiment using dephosphorylated plasmid
pNS:LY7.
Characterization of bacteria containing the 25 mer
leader pNS:LY7 (gln)
Colonies of E. coli were picked and incubat~d
onto two sheets of nitrocellulose filter spread on YT
plate with ampicillin. A master plate was also prepared.
The filter is first soaked with o.5 N NaOH + 1.5 N NaCl
soln and then neutralized with 0.5 N Tris-HCl (pH 7) +
1.5 N NaCl. Filters were baked in a vacuum oven at 80
for 2 hr. After being washed in 6X sodium chloride/
sodium citrate solution (SSC) + 0.05% Triton X-100,
cell debris was removed from filters and the filters
were washed with a prehybridization mixture of 6X S
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1% dextran sulfate, lX Denhardt solution and 0.05%
Triton X-100 for 20 min. A probe solution, prepared
by treating 10 pmole of 25-mer probe, with 1 ~1 lOX
kinase buffer, 1 ~1 kinase and 10 pmole of 32p ATP
(3 ~1) at 37 for 1 hr, was added. After 16 hr at 45,
the filter was washed twice with 15 ml of 6X Ssc and
0.05% Triton X-100 at room temperature for 5 min and
then once at 45 for 30 min. The filter was then
exposed to X-ray film. Generally in 40 colonies, 5 may
have multiple copies of 25 mer leader and 30 would have a
single copy of the insert. The plasmid was then prepared
with the procedure suggested by BRL and cut by Hae III
for dideoxy DNA sequenciny.
Induction of proinsulin
An overnight grown culture of E. coli (JM103)
containing plasmid pNS:LY7 (gln) in 2YT + ampicillin
was diluted with 2YT medium + ampicillin (8 ml) in a
ratio of 1:100. After 2 hr. at 37 , IPTG was added to
a final concentration of 0.7 mM. After 24 hr, cells
were collected by centrifugation at 5000 rev/min for
10 min. The cell pellet was suspended in 2 ml of 6M
guanidine HCl (pH 7) or 1% SDS solution. After lysis
of the cell via sonication, cell debris was removed
by centrifugation at 6000 rev/min for 10 min. The
solution was analyzed by the radioimmunoassay method
13~1620~3
for both C-peptide and insulin activity as described
below.
Radioimmunoassay for C-peptide
The cell content solution (guanidine-HCl)
was assayed for human proinsulin by the human [125I]tyro-
~ 0 1~
sine C-peptide antibody kit (~n~i Industri) with
synthetic C-peptide as the standard. The amount of
proinsulin was estimated on the suggestion by the
manufacturer that reactivity of proinsulin towards the
anti serum is 11~ of that of synthetic C-peptide on
an equal molar basis. The cell content solution of
pNS:LY7 ~gln, cys and ser) had to be diluted lO00 times
before it can be determined by an RIA test.
Radioimmunoassay for insulin
The cell content solution was also tested
for insulin (AB chains) antigenic activity by an RIA
kit supplied by Amersham.
Characterization of fused protein
The cell content solution (1% SDS) was also
analyzed by Laemmli's 15~ SDS-polyacrylamide gel. It
is either stained with Coomassie blue or silver nitrate.
For characterization of the fused protein,
a protein gel of cell solution of pNS:LY7 (gln) was
cut in fourteen sections which were then eluted with
1~ SDS solution. The eluent was then tested by the
C-peptide RIA method.
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Chemical cleavage of fused protein into proinsulin
Fused product was purified by a preparative
15% SDS-polyacrylamide gel, and was then treated with
50 mole equivalent of cyanogen bromide in 70% formic
acid per mg of protein for 24 hr at room temperature.
The product was then analyzed with 15% SDS-polyacrylamide
gel.
Table I. Production of hybrid proinsulin
Leader CloneEstimated proinsulin yield
mg/Q culture
with IPTG without IPTG
-gln- pNS:LY7 (gln)146 56
-cys- ,pNS:LY7 ~cys) 75
-ser- pNS:LY7 (ser)140
-leu- pNS:LY7 ~leu)0.2
control 0.0
(JM103)
In 1 litre of culture medium, one can obtain 1.0 gm of
total bacterial protein; therefore pNS:LY7 (gln) under
the effect of IPTG has 10-15% of its bacterial protein
as proinsulin.
Table I shows that homo-amino acid
leaders, the amino acid being selected from
glutamine, cysteine and serine produce good results in
conjunction with human proinsulin. However if the
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` 1306~:0~3
homo-amino acid is leucine results are poor indicating
that a match of the selected leader signal sequence and
the sequence coding for a desired polypeptide needs to
be made (see also Figure 1).
It should be emphasised that this invention
is distinct from S.A. Narang et al Can. Patent Application
483,100 of 4 June 1985. Narang et al, in this Can.
application, are concerned with affinity leader sequences
which aid purification by attachment of the combined
polypeptide, coded by the DNA sequence, to affinity column
media. An example given therein concerns the use of an
affinity leader sequence coding for a homo-amino acid
sequence such as poly glutamine in combination with a
human preproinsulin coding gene. We have found that such
a combination does not show enhanced expression and
stabilization of the gene product of the magnitude shown
here. It appears that the "pre" part of the preproinsulin
counteracts such an effect of such a leader and that this
enhanced expression and stabilization is an interactive
property of the composite polypeptide product of such
a DNA sequence (for example, the polyglutamine
proinsulin reported here) rather than a property of
one part of such a polypeptide product that can be
added on with no effect on the other properties of the
composite polypeptide. However, in one of the examples
~7
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reported here, the example employing polycysteine as a
pol~lpeptide leader, the polycysteine leader has both
the useful functional properties of enhancing effective
expression and stabilizing the composite polypeptide,
when the other component of such a composite polypeptide
is proinsulin, and is recognised and bound by affinity
column media. This combination of increased effective
yield and ease of purification should prove most useful
in the production of polypeptides by microorganisms.
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