GB2039916A - Protein synthesis by genetic manipulation - Google Patents
Protein synthesis by genetic manipulation Download PDFInfo
- Publication number
- GB2039916A GB2039916A GB8001106A GB8001106A GB2039916A GB 2039916 A GB2039916 A GB 2039916A GB 8001106 A GB8001106 A GB 8001106A GB 8001106 A GB8001106 A GB 8001106A GB 2039916 A GB2039916 A GB 2039916A
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- GB
- United Kingdom
- Prior art keywords
- gene
- site
- prokaryotic
- bacteria
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- 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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- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
- C12N15/72—Expression systems using regulatory sequences derived from the lac-operon
-
- 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
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A process to produce specific proteins coded for by eukaryotic (or prokaryotic) DNA in bacteria. The process which uses recombinant DNA techniques, produces proteins in their natural, functional state unencumbered by extraneous peptides.
Description
SPECIFICATION
Protein synthesis
This invention is a method for producing in bacteria prokaryotic or eukaryotic proteins in native, unfused form free from extraneous peptides.
Recombinant DNA techniques in vitro have been used to insert a variety of eukaryotic genes into plasmids carried by Escherichia coli in an effort to induce these bacteria to produce eukaryotic proteins. Most of these genes have not directed the synthesis of the native proteins because the eukaryotic signals coding for initiation of transcription and/or translation do not function well in E. coli. One proposed solution to this problem has been the fusion of the eukaryotic gene with a bacterial gene.
The process results in the production of a hybrid protein, a portion of which at its carboxyl terminus is constituted by the eukaryotic protein. In one case, it has been possible to separate a small biologically active protein from a fusion product (Itakura,
K. et al., Science 198, 1056 (1977)).
Gene expression takes place by transcription into mRNA then translation into protein. To do these operations, the DNA preceding the gene must have a sequence which: (a) directs efficient binding of bacterial RNA polymerase and efficient initiation of transcription, and (B) codes for a mRNA that directs efficient binding of mRNA to the ribosomes and initiation of translation into protein.
The present invention provides a method of producing native, unfused prokaryotic or eukaryotic protein in bacteria which comprises inserting into a bacterial plasmid a gene for a prokaryotic or eukaryotic protein and a portable promotor consisting of a
DNA fragment containing a transcription initiation site recognized by RNA polymerase and containing no protein translation start site, said promotor being inserted ahead of a protein translational start site of said gene to form a fused gene having a hybrid ribosome binding site, inserting said plasmid into said bacteria to transform said bacteria with said plasmid containing said fused gene, and culturing the transformed bacteria to produce said prokaryotic or eukaryotic protein.
The present invention utilizes nucleases, restriction enzymes, and DNA ligase to position a portable promoter consisting of a DNA fragment containing a transcription site but no translation initiation site nearthe beginning ofthe gene which codesforthe desired protein to form a hybrid ribosomal binding site. The protein produced by the bacterium from this hybrid is the native derivative of the implanted gene. It has been found that the endonuclease digestion product of the E. colt lay operon, a fragment of DNA which contains a transcription initiation site but no translational start site, has the required properties to function as a portable promoter in the present invention, being transcribed at high efficiency by bacterial RNA polymerase.The mRNA produced contains a ribosomal binding site (Shine-Dalgerno (S-D) Sequence) but it does not include the AUG or GUG required for translational initiation. However, in accordance with the present invention, a hybrid is formed consisting of the S-D sequence and initiator from the lac operon and the
AUG sequence of the gene, and such a fused gene is translated and transcribed efficiently. Using the enzymes exonuclease Ill and So, the promotor may be put at any desired position in front of the translational start site of the gene in order to obtain optimum production of protein.Since the promotor can be inserted at a restriction site ahead of the translational start site of the gene, the gene can first be cut at the restriction site, the desired number of base pairs and any single stranded tails can be removed by treating with nucleases for the appropriate time period, and religating.
The following specific example is intended to illustrate more fully the nature of the present invention without acting as a limitation upon its scope.
Example
A rabbit P-globin gene was first cloned into the Hin
Ill site of pBR322, a plasmid of the E. coli bacteria, via restriction enzyme cuts of the initial DNA, reconstitution of the gene by T4 ligase, insertion of the reconstituted gene into the Hin Ill site using chemically synthesized Hin Ill linkers, and religating with
DNA ligase.
The Hin Ill cut at the carboxyl end of the cloned gene was removed by partialiy digesting with Hin III, filling in the resulting Hin III "sticky ends" with E. coli
DNA polymerase I, and religating with T4 ligase. This left in the resulting plasmid a single Hin III cut 25 base pairs ahead of the amino terminus of the globin gene.
Differing numbers of the 25 base pairs between the Hin III cut and the ATG signalling the start point of translation were removed from different samples of the cloned gene as follows: the plasmid was cut with Hin Ill, resected for various times from 0.5 to 10 minutes with Exo Ill, then treated with SI to remove single-stranded tails.
The portable promoter of the lac operon, an
R1-Alu restriction fragment of E. coli DNA, was then inserted by treating each sample of the plasmid with
RI which cuts at a unique site some 30 base pairs upstream from the Hin Ill site, and the portable promotor was inserted into the plasmid backbone at this site. This requires one "sticky end" and one "flush" end, both of which are ligated by the same treatment with ligase.
Colonies of E. coli each containing one of these resulting plasmids were then screened for (3-globin production using RIA-screening techniques to identify the one or more producing p-globin.
The globin gene in the above construction can be any gene coding for prokaryotic or eukaryotic proteins, and any other unique restriction site can be employed in place of the Hin Ill site. If the restriction site is located inconveniently far from the beginning of the gene, it may be moved (for example, a Hin Ill site may be moved by opening the plasmid with Hin
Ill, digesting with Exo Ill and S1,then religating the resulting plasmid in the presence of excess Hin Ill linkers). Any suitable restriction site can be employed for insertion of the portable promotor in place of the R1 site (e.g. Pst, BAM, or Sal I). Finally, it should be emphasized that the most difficult step, the cloning of the gene into the plasmid, is done once and left unchanged. The promotor fragment will confer its constitutive expression on the cell so it is easy to screen for the intact promoters.
Claims (10)
1. A fused gene coding for a hybrid ribosome binding site comprising
the region of a bacterial gene comprising, inorder of transcription,
the SD region, and
at least one base pair adjacent to said SD region, fused to
the region of a gene for a prokaryotic or eukaryotic protein comprising, in order of transcription,
at least one base pair adjacent to a translation start site, and
said translation start site.
2. The fused gene of claim 1, further comprising
ahead of said sd region, the promotor region of said bacterial gene, and
after said translation start site of said gene for said prokaryotic or eukaryotic protein, the region of said gene coding for said prokaryotic or eukaryotic protein.
3. The method of producing native, unfused prokaryotic or eukaryotic protein in bacteria which comprises inserting into a bacterial plasmid a gene for a prokaryotic or eukaryotic protein and a portable promotor consisting of a DNA fragment containing a transcription initiation site recognized by RNA polymerase and containing no protein translational start site, said promoter being inserted ahead of a protein translational start site of said gene to form a fused gene having a hybrid ribosome binding site, inserting said plasmid into said bacteria to transform said bacteria with said plasmid containing said fused gene, and culturing the transformed bacteria to produce said prokaryotic or eukaryotic protein.
4. The method as claimed in claim 3 in which said bacteria is E. coli.
5. The method as claimed in claims 3 or 4 in which said portable promotor is the product of restriction endonuclease digestion of an operon.
6. The method claimed in claim 4 in which said portable promotor is the product of restriction endonuclease digestion ofthelacoperon of E. coli.
7. The method as claimed in claims 3 or 4 in which said gene insertion comprises cloning said gene into said plasmid, adjusting the spacing between said inserted gene and a preceding unique restriction site by treating with a nuclease, and cloning said portable promotor into said restriction site.
8. The method as claimed in claim 4 in which said gene insertion comprises cloning said gene into said plasmid, adjusting the spacing between said gene and a preceding unique restriction site by treating with a nuclease, and cloning into said restriction site a portable promoter formed by the endonuclease digestion ofthelacoperon of E. coli.
9. A fused gene coding for a hybrid ribosome site substantially as hereinbefore described with reference to the Example.
10. A method of producing native, unfused prokaryotic or eukaryotic protein in bacteria substantially as hereinbefore described with reference to the < Example.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US310279A | 1979-01-15 | 1979-01-15 |
Publications (1)
Publication Number | Publication Date |
---|---|
GB2039916A true GB2039916A (en) | 1980-08-20 |
Family
ID=21704160
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB8001106A Withdrawn GB2039916A (en) | 1979-01-15 | 1980-01-14 | Protein synthesis by genetic manipulation |
Country Status (7)
Country | Link |
---|---|
JP (1) | JPS5599193A (en) |
CA (1) | CA1174617A (en) |
DE (1) | DE3000982A1 (en) |
FR (1) | FR2446318A1 (en) |
GB (1) | GB2039916A (en) |
NL (1) | NL8000127A (en) |
SE (1) | SE8000297L (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0063494A2 (en) * | 1981-04-20 | 1982-10-27 | Cetus Corporation | Method for producing protein from a microorganism, microorganisms for use in such method and creation thereof, vectors for use in said creation, and protein produced thereby, and transformant culture derived from said microorganisms |
WO1985003522A1 (en) * | 1984-02-08 | 1985-08-15 | Cetus Corporation | Monitoring and control systems for recombinant manipulations |
US4874702A (en) * | 1980-09-08 | 1989-10-17 | Biogen, Inc. | Vectors and methods for making such vectors and for expressive cloned genes |
US5860189A (en) * | 1997-03-06 | 1999-01-19 | An; Tae-Heup | Door wheel |
US7588755B1 (en) | 1980-04-03 | 2009-09-15 | Biogen Idec Ma Inc. | DNA sequences, recombinant DNA molecules and processes for producing human fibroblast interferon-like polypeptides |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IT1144705B (en) * | 1980-03-17 | 1986-10-29 | Harvard College | OPTIMAL PRODUCTION OF POLYPEPTIDES WITH THE USE OF FUSIONATED GENES |
US4582800A (en) * | 1982-07-12 | 1986-04-15 | Hoffmann-La Roche Inc. | Novel vectors and method for controlling interferon expression |
JPS5965099A (en) * | 1982-10-05 | 1984-04-13 | Takeda Chem Ind Ltd | Promoter for expression and its use |
-
1980
- 1980-01-09 NL NL8000127A patent/NL8000127A/en not_active Application Discontinuation
- 1980-01-12 DE DE19803000982 patent/DE3000982A1/en not_active Withdrawn
- 1980-01-14 SE SE8000297A patent/SE8000297L/en unknown
- 1980-01-14 GB GB8001106A patent/GB2039916A/en not_active Withdrawn
- 1980-01-14 CA CA000343606A patent/CA1174617A/en not_active Expired
- 1980-01-15 FR FR8000848A patent/FR2446318A1/en not_active Withdrawn
- 1980-01-16 JP JP346580A patent/JPS5599193A/en active Pending
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7588755B1 (en) | 1980-04-03 | 2009-09-15 | Biogen Idec Ma Inc. | DNA sequences, recombinant DNA molecules and processes for producing human fibroblast interferon-like polypeptides |
US7635466B1 (en) | 1980-04-03 | 2009-12-22 | Biogen Idec Ma Inc. | DNA sequences, recombinant DNA molecules and processes for producing human fibroblast interferon-like polypeptides |
US4874702A (en) * | 1980-09-08 | 1989-10-17 | Biogen, Inc. | Vectors and methods for making such vectors and for expressive cloned genes |
EP0063494A2 (en) * | 1981-04-20 | 1982-10-27 | Cetus Corporation | Method for producing protein from a microorganism, microorganisms for use in such method and creation thereof, vectors for use in said creation, and protein produced thereby, and transformant culture derived from said microorganisms |
EP0063494A3 (en) * | 1981-04-20 | 1984-02-22 | Cetus Corporation | Method for producing protein from a microorganism, microorganisms for use in such method and creation thereof, vectors for use in said creation, and protein produced thereby, and transformant culture derived from said microorganisms |
WO1985003522A1 (en) * | 1984-02-08 | 1985-08-15 | Cetus Corporation | Monitoring and control systems for recombinant manipulations |
US5860189A (en) * | 1997-03-06 | 1999-01-19 | An; Tae-Heup | Door wheel |
Also Published As
Publication number | Publication date |
---|---|
NL8000127A (en) | 1980-07-17 |
FR2446318A1 (en) | 1980-08-08 |
JPS5599193A (en) | 1980-07-28 |
SE8000297L (en) | 1980-09-08 |
CA1174617A (en) | 1984-09-18 |
DE3000982A1 (en) | 1980-08-07 |
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Legal Events
Date | Code | Title | Description |
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WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |