CA1306208C - Enhanced expression and stabilization of gene products in microorganisms using short selected leader signal sequences - Google Patents
Enhanced expression and stabilization of gene products in microorganisms using short selected leader signal sequencesInfo
- Publication number
- CA1306208C CA1306208C CA 471799 CA471799A CA1306208C CA 1306208 C CA1306208 C CA 1306208C CA 471799 CA471799 CA 471799 CA 471799 A CA471799 A CA 471799A CA 1306208 C CA1306208 C CA 1306208C
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- CA
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- Prior art keywords
- sequence
- polypeptide
- dna sequence
- codes
- peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
Abstract
TITLE
ENHANCED EXPRESSION AND STABILIZATION OF GENE PRODUCTS IN
MICROORGANISMS USING SHORT SELECTED LEADER SIGNAL SEQUENCES
INVENTORS
Saran A. Narang Winq L. Sung ABSTRACT
This invention concerns the employment of DNA
sequences comprising (a) a sequence coding for a desired polypeptide, and; (b) a selected leader signal sequence coding for a range of from a tri-peptide to about one third of the size of peptide coded by sequence (a), said selected leader signal sequence being chosen to enhance the effective expression of, and/or stabilize the poly-peptide coded by sequences (a) and (b) when said DNA
sequence resides and is expressed in a foreign host microorganism. The polypeptide products of sequences (a) and (b) preferably should be readily cleavable from each other to allow for simple purification of such products.
ENHANCED EXPRESSION AND STABILIZATION OF GENE PRODUCTS IN
MICROORGANISMS USING SHORT SELECTED LEADER SIGNAL SEQUENCES
INVENTORS
Saran A. Narang Winq L. Sung ABSTRACT
This invention concerns the employment of DNA
sequences comprising (a) a sequence coding for a desired polypeptide, and; (b) a selected leader signal sequence coding for a range of from a tri-peptide to about one third of the size of peptide coded by sequence (a), said selected leader signal sequence being chosen to enhance the effective expression of, and/or stabilize the poly-peptide coded by sequences (a) and (b) when said DNA
sequence resides and is expressed in a foreign host microorganism. The polypeptide products of sequences (a) and (b) preferably should be readily cleavable from each other to allow for simple purification of such products.
Description
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 , ~
.. .
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 . _ . , 1306~08 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.
~ ' -'', . ` ",, ,', ' , . ', ', ` i;~0620~
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 5 _ .
---" 13Q62013 `
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O ~~ O
."C,~ .,.
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C~ ~ . .
'SJ ~ ' tC~~ ~ ~
nCl~ <~ J
C~ ~~ C~ t ~ ~ I_ ) ~ ~ _ ~ J
aJ ~ 2 L~ ~ J J ~ CL
~) f ~ ~ ~ ~
Z aJ ~--~1 ~ CC
C~ ~
U~ J
Q
L ~ llt ~:
C ~ C
v~
_ lV O L ~ L
~ . ~ _ _ _ /~ a v~ zc J
l ~ Z ~- L ~ ~ C~
Z I
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'S
CC Cl~
, , ~3Q6~08 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
13~6~08 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.
I3(~6~0~
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 ` 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 13062Q~
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.
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 , ~
.. .
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 . _ . , 1306~08 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.
~ ' -'', . ` ",, ,', ' , . ', ', ` i;~0620~
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 5 _ .
---" 13Q62013 `
C C
.~ . .
C :~ ~ C ~ ~
.. .. . .
O ~~ O
."C,~ .,.
S_~ t(,~
CC C
J~:
C~ ~ . .
'SJ ~ ' tC~~ ~ ~
nCl~ <~ J
C~ ~~ C~ t ~ ~ I_ ) ~ ~ _ ~ J
aJ ~ 2 L~ ~ J J ~ CL
~) f ~ ~ ~ ~
Z aJ ~--~1 ~ CC
C~ ~
U~ J
Q
L ~ llt ~:
C ~ C
v~
_ lV O L ~ L
~ . ~ _ _ _ /~ a v~ zc J
l ~ Z ~- L ~ ~ C~
Z I
J ~ ~ ~
'S
CC Cl~
, , ~3Q6~08 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
13~6~08 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.
I3(~6~0~
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 ` 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 13062Q~
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.
Claims (16)
1. A DNA sequence comprising:
(a) a DNA sequence component coding for a desired polypeptide; and (b) a selected leader DNA sequence component coding for a range of from a tri-peptide to about one third of the size of peptide coded by sequence (a); said selected leader sequence coding for an amino acid sequence chosen to enhance the effective expression of, and/or stabilize the polypeptide coded by sequences (a) and (b) when said DNA sequence (a)+(b) resides and is expressed in a foreign host cell.
(a) a DNA sequence component coding for a desired polypeptide; and (b) a selected leader DNA sequence component coding for a range of from a tri-peptide to about one third of the size of peptide coded by sequence (a); said selected leader sequence coding for an amino acid sequence chosen to enhance the effective expression of, and/or stabilize the polypeptide coded by sequences (a) and (b) when said DNA sequence (a)+(b) resides and is expressed in a foreign host cell.
2. The DNA sequence of claim 1 wherein (b) codes for a peptide 5-10 amino acids in length.
3. The DNA sequence of claim 1 wherein (b) codes for a peptide having amino acids selected from glutamine, cysteine and serine.
4. The DNA sequence of claims 1, 2 or 3 wherein (b) codes for a homo-amino acid sequence.
5. The DNA sequence of claims 1, 2 or 3 wherein (a) codes for one of insulin, proinsulin, somatostatin or one of the interferons.
6. The DNA sequence of claims 1, 2 or 3 wherein (a) codes for human proinsulin.
7. The DNA sequence of claim 1 wherein (a) codes for human proinsulin and (b) codes for a homo-amino acid sequence 5-10 residues long.
CLAIMS (cont.)
CLAIMS (cont.)
8. The DNA sequence of claims 1, 2 or 3 in a clon-ing vehicle, wherein said vehicle is a plasmid of the PNS:LY7 type.
9. The DNA sequence of claims 1, 2 or 3 wherein sequence (a) is devoid of any pre sequence.
10. An E. coli microorganism containing the DNA
sequence of claims 1, 2 or 3.
sequence of claims 1, 2 or 3.
11. E. coli host cells containing the DNA sequence of claim 7.
12. A method of making a modified host cell capable of producing an increased yield of a desired foreign polypeptide comprising:
(i) ligating a DNA sequence coding for the desired polypeptide (sequence (a)) to an opened selected cloning vehicle sequence;
(ii) ligating, to the DNA sequence resulting from step (i) upstream of sequence (a), a selected leader 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), wherein said selected leader sequence is an amino acid sequence selected to enhance the expression of, and/or stabilize the polypeptide coded by sequence (a)+(b) when expressed in said host cell;
(iii) placing the cloning vehicle resulting from step (ii) in a selected host cell and recovering the modified host cell.
CLAIMS (cont.)
(i) ligating a DNA sequence coding for the desired polypeptide (sequence (a)) to an opened selected cloning vehicle sequence;
(ii) ligating, to the DNA sequence resulting from step (i) upstream of sequence (a), a selected leader 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), wherein said selected leader sequence is an amino acid sequence selected to enhance the expression of, and/or stabilize the polypeptide coded by sequence (a)+(b) when expressed in said host cell;
(iii) placing the cloning vehicle resulting from step (ii) in a selected host cell and recovering the modified host cell.
CLAIMS (cont.)
13. The method of claim 12 wherein the host cell is E. coli, sequence (a) codes for human proinsulin, and sequence (b) codes for a homo-amino acid sequence 5-10 residues long.
14. A biological method of producing a desired polypeptide comprising:
(iv) culturing modified host cells resulting from step (iii) of claim 12 in conditions that allow for the expression of the gene coding for the desired poly-peptide;
(v) isolating the polypeptide coded by sequences (a)+(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) purifying the product of step (vi) to yield the desired sequence (a) polypeptide in amounts increased over those obtained in the absence of the selected leader sequence.
(iv) culturing modified host cells resulting from step (iii) of claim 12 in conditions that allow for the expression of the gene coding for the desired poly-peptide;
(v) isolating the polypeptide coded by sequences (a)+(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) purifying the product of step (vi) to yield the desired sequence (a) polypeptide in amounts increased over those obtained in the absence of the selected leader sequence.
15. The method of claim 14 wherein the desired polypeptide is human proinsulin, the host cells are E.
coli, sequence (b) codes for a homo-amino acid sequence 5-10 residues long and step (vi) comprises cyanogen bro-mide cleavage.
coli, sequence (b) codes for a homo-amino acid sequence 5-10 residues long and step (vi) comprises cyanogen bro-mide cleavage.
16. The method of claim 12, 14 or 15 wherein the sequence (a) is devoid of any pre sequence.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 471799 CA1306208C (en) | 1985-01-09 | 1985-01-09 | Enhanced expression and stabilization of gene products in microorganisms using short selected leader signal sequences |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 471799 CA1306208C (en) | 1985-01-09 | 1985-01-09 | Enhanced expression and stabilization of gene products in microorganisms using short selected leader signal sequences |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1306208C true CA1306208C (en) | 1992-08-11 |
Family
ID=4129550
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 471799 Expired - Fee Related CA1306208C (en) | 1985-01-09 | 1985-01-09 | Enhanced expression and stabilization of gene products in microorganisms using short selected leader signal sequences |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1306208C (en) |
-
1985
- 1985-01-09 CA CA 471799 patent/CA1306208C/en not_active Expired - Fee Related
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