CA1174617A - Protein synthesis - Google Patents
Protein synthesisInfo
- Publication number
- CA1174617A CA1174617A CA000343606A CA343606A CA1174617A CA 1174617 A CA1174617 A CA 1174617A CA 000343606 A CA000343606 A CA 000343606A CA 343606 A CA343606 A CA 343606A CA 1174617 A CA1174617 A CA 1174617A
- Authority
- CA
- Canada
- Prior art keywords
- gene
- site
- protein
- prokaryotic
- promoter
- 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.)
- Expired
Links
Classifications
-
- 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
Landscapes
- 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
Abstract of the Disclosure This invention is a produce to produce specific proteins coded for by eukaryotic (or prokaryotic) DNA in bacteria. The invention, which uses recombinant DNA techniques, produces proteins in their natural, function state unencumberd by extraneous peptides.
Description
~:1 746~7 The invention described herein was made in the course of work under a grant or award from the National Science Foundation.
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 proeess results in the produetion of a hybrid protein, a portion of whieh at its earboxyl terminus is eonstituted 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 ~f translation into protein.
The present invention provides a method of producing native, unfused prokaryotic or eukaryotic protein in bacteria which comprises ~ ~746~7 inserting into a bactexial plasmid a gene for a prokaryotic or eukaryotic protein and a portable promoter consisting of a DNA
fragment containing a Shine-Dalgarno sequence and a transcription initiation site recognized by RNA polymer~se and containing no protein translational start site, said promoter being inserted upstream from a protein ATG translational start site of said gene at a position to obtain production of said protein, to form a fused gene having said Shine-Dalgarno sequence, said transcription initiation site, and the ATG signalling the start of translation, inserting said plasmid into said bacteria to transform said bact,eria with said plasmid containing said fused gene, and cultur-ing the transformed bacteria to produce said unfused prokaryotic or eukaryotic protein.
In another aspect, the present invention provides a fused gene capable of expressing native unfused prokaryotic or eukaryotic protein comprising (1) a portable promoter including a portion of a bacterial gene having a Shine-Dalgarno sequence and a transcription initiation site recognized by RNA polymerase and containing no protein translational start site, and (2) fused thereto a gene for a native unfused prokaryotic or eukaryotic protein including its translational start site, said portable promoter being located upstream from said translational start site.
In a further aspect, the present invention provides a bacterium containing the fused gene of alaim ~ within a bacterial plasmid said bacterium being capable of producing said prokaryotic or said eukaryotic protein.
The present invention utilizes nucleases, restriction enzymes, and DNA ligase to position a portable promoter consisting 1:17~161~
of a DNA fragment containing a transcription site but no transla-tion initiation site near the begi,nning of the gene which codes for the 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. coli lac 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-Dalgarno-(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 ATG sequence of the gene, and such a fused gene is translated and transcribed efficiently.
- 2a -~ ~7~6i17 Using the enzymes exonuclease III a~d Sl, the promoter 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 promoter 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 B-globin gene was first cloned into the Hin III 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 III site using chemically synthesized Hin III
linkers, and religating with DNA ligase.
The Hin III cut at the carboxyl end of the cloned gene was removed by partially 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 termimus 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 coloned gene as follows: the plasmid was cut with Hin III, resected for various times from 0.5 to 10 minutes with Exo III, then treated with Sl to remove single-stranded tàilS.
~:174~
The portable promoter of the lac operon, an Rl-Alu restriction fragment of E. coli DNA, was then inserted by -treating each sample of the plasmid with Rl which cuts at a unique site some 30 base pairs upstream from the Hin III site, and the portable promoter 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 ~. coli each containing one of these resulting plasmids were then screened for B-globin production using RIA-screening techniques to identify the one or more producing B-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 III site. If the restriction site is located inconveniently far from the beginning of the gene, it may be moved (for example, a Hin III site may be moved by opening the plasmid with Hin III, digesting with Exo III and SI, then religating the resulting plasmid in the presence of excess Hin III linkers). Any suitable restriction site can be employed for insertion of the portable promoter in place of the Rl site (e.g. Pst, BAM, or Sal I). Finally, it should be emphasiæed that the most difficult step, the cloning of the gene into the plasmid, is done once and left unchanged. The promoter fragment will confer its constitutive expression on the cell so it is easy to screen for the intact promoters.
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 proeess results in the produetion of a hybrid protein, a portion of whieh at its earboxyl terminus is eonstituted 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 ~f translation into protein.
The present invention provides a method of producing native, unfused prokaryotic or eukaryotic protein in bacteria which comprises ~ ~746~7 inserting into a bactexial plasmid a gene for a prokaryotic or eukaryotic protein and a portable promoter consisting of a DNA
fragment containing a Shine-Dalgarno sequence and a transcription initiation site recognized by RNA polymer~se and containing no protein translational start site, said promoter being inserted upstream from a protein ATG translational start site of said gene at a position to obtain production of said protein, to form a fused gene having said Shine-Dalgarno sequence, said transcription initiation site, and the ATG signalling the start of translation, inserting said plasmid into said bacteria to transform said bact,eria with said plasmid containing said fused gene, and cultur-ing the transformed bacteria to produce said unfused prokaryotic or eukaryotic protein.
In another aspect, the present invention provides a fused gene capable of expressing native unfused prokaryotic or eukaryotic protein comprising (1) a portable promoter including a portion of a bacterial gene having a Shine-Dalgarno sequence and a transcription initiation site recognized by RNA polymerase and containing no protein translational start site, and (2) fused thereto a gene for a native unfused prokaryotic or eukaryotic protein including its translational start site, said portable promoter being located upstream from said translational start site.
In a further aspect, the present invention provides a bacterium containing the fused gene of alaim ~ within a bacterial plasmid said bacterium being capable of producing said prokaryotic or said eukaryotic protein.
The present invention utilizes nucleases, restriction enzymes, and DNA ligase to position a portable promoter consisting 1:17~161~
of a DNA fragment containing a transcription site but no transla-tion initiation site near the begi,nning of the gene which codes for the 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. coli lac 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-Dalgarno-(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 ATG sequence of the gene, and such a fused gene is translated and transcribed efficiently.
- 2a -~ ~7~6i17 Using the enzymes exonuclease III a~d Sl, the promoter 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 promoter 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 B-globin gene was first cloned into the Hin III 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 III site using chemically synthesized Hin III
linkers, and religating with DNA ligase.
The Hin III cut at the carboxyl end of the cloned gene was removed by partially 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 termimus 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 coloned gene as follows: the plasmid was cut with Hin III, resected for various times from 0.5 to 10 minutes with Exo III, then treated with Sl to remove single-stranded tàilS.
~:174~
The portable promoter of the lac operon, an Rl-Alu restriction fragment of E. coli DNA, was then inserted by -treating each sample of the plasmid with Rl which cuts at a unique site some 30 base pairs upstream from the Hin III site, and the portable promoter 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 ~. coli each containing one of these resulting plasmids were then screened for B-globin production using RIA-screening techniques to identify the one or more producing B-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 III site. If the restriction site is located inconveniently far from the beginning of the gene, it may be moved (for example, a Hin III site may be moved by opening the plasmid with Hin III, digesting with Exo III and SI, then religating the resulting plasmid in the presence of excess Hin III linkers). Any suitable restriction site can be employed for insertion of the portable promoter in place of the Rl site (e.g. Pst, BAM, or Sal I). Finally, it should be emphasiæed that the most difficult step, the cloning of the gene into the plasmid, is done once and left unchanged. The promoter fragment will confer its constitutive expression on the cell so it is easy to screen for the intact promoters.
Claims (14)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of producing native, unfused prokaryotic or eukaryotic protein in bacteria which comprises inserting into a bacterial plasmid a gene for a pro-karyotic or eukaryotic protein and a portable promoter consisting of a DNA fragment containing a Shine-Dalgarno sequence and a transcription initiation site recognized by RNA polymerase and containing no protein translational start site, said promoter being inserted upstream from a protein ATG translational start site of said gene, at a position to obtain production of said protein, to form a fused gene having said Shine-Dalgarno sequence, said transcription initiation site, and the ATG signalling the start point of translation, 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 unfused prokaryotic or eukaryotic protein.
2. The method as claimed in claim 1 in which said bacteria is E. coli.
3. The method as claimed in claims 1 or 2 in which said portable promoter is the product of restriction endonulcease digestion of an operon.
4. The method claimed in claim 2 in which said portable promoter is the product of restriction endonuclease digestion of the lac operon of E. coli.
5. The method as claimed in claims 1 or 2 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 promoter into said restriction site.
6. The method as claimed in claim 2 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 of the lac operon of E. coli.
7. A fused gene capable of expressing native unfused prokaryotic or eukaryotic protein comprising (1) a portable promoter including a portion of a bacterial gene having a Shine-Dalgarno sequence and a transcription initiation site recognized by RNA polymerase and containing no protein translation-al start site, and (2) fused thereot a gene for a native unfused prokaryotic or eukaryotic protein including its translational start site, said portable promoter being located upstream from said translational start site.
8. A fused gene according to claim 7 in which said promoter is the product of restriction endonuclease digestion of an operon.
9. A fused gene according to claim 7 in which said bacterial gene is that of E. coli and said promoter is the product of res-triction endonuclease digestion of the lac operon of E. coli.
10. A fused gene according to claim 7 wherein said translational start site of said gene for said prokaryotic or eukaryotic protein is the sequence ATG.
11. A fused gene according to claim 7 wherein said portable promoter and said translational start site of said prokaryotic or eukaryotic gene together comprise a hybrid ribosomal binding site.
12. A fused gene according to claim 7 wherein said gene for a prokaryotic or eukaryotic protein is a gene for a eukaryotic protein.
13. A bacterium containing the fused gene of claim 7 within a bacterial plasmid, said bacterium being capable of producing said prokaryotic or said eukaryotic protein.
14. A bacterium containing the fused gene of claim 12 within a bacterial plasmid said bacterium being capable of producing said eukaryotic protein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US310279A | 1979-01-15 | 1979-01-15 | |
| US3,102 | 1987-01-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1174617A true CA1174617A (en) | 1984-09-18 |
Family
ID=21704160
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000343606A Expired CA1174617A (en) | 1979-01-15 | 1980-01-14 | Protein synthesis |
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) |
Families Citing this family (8)
| 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 |
| NZ196706A (en) | 1980-04-03 | 1988-01-08 | Biogen Nv | Dna sequences, recombinant dna molecules and processes for producing fibroblast interferon-like polypeptides; pharmaceutical and veterinary compositions |
| US4874702A (en) * | 1980-09-08 | 1989-10-17 | Biogen, Inc. | Vectors and methods for making such vectors and for expressive cloned genes |
| CA1204681A (en) * | 1981-04-20 | 1986-05-20 | Cetus Corporation | Method and vector organism for controlled accumulation of cloned heterologous gene products in bacillus subtilis ii |
| 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 |
| ATE100494T1 (en) * | 1984-02-08 | 1994-02-15 | Cetus Oncology Corp | CONTROL SYSTEMS FOR RECOMBINANT MANIPULATIONS. |
| US5860189A (en) * | 1997-03-06 | 1999-01-19 | An; Tae-Heup | Door wheel |
-
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 CA CA000343606A patent/CA1174617A/en not_active Expired
- 1980-01-14 GB GB8001106A patent/GB2039916A/en not_active Withdrawn
- 1980-01-15 FR FR8000848A patent/FR2446318A1/en not_active Withdrawn
- 1980-01-16 JP JP346580A patent/JPS5599193A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5599193A (en) | 1980-07-28 |
| NL8000127A (en) | 1980-07-17 |
| GB2039916A (en) | 1980-08-20 |
| SE8000297L (en) | 1980-09-08 |
| DE3000982A1 (en) | 1980-08-07 |
| FR2446318A1 (en) | 1980-08-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4332892A (en) | Protein synthesis | |
| US4418149A (en) | Fused hybrid gene | |
| EP0267851A2 (en) | Hybrid promoter, expression controlling DNA sequence and expression vector | |
| CA1318867C (en) | Plasmid expression vector for use in escherichia coli | |
| US4663290A (en) | Production of reverse transcriptase | |
| US4536475A (en) | Plant vector | |
| Genilloud et al. | The transposon Tn5 carries a bleomycin-resistance determinant | |
| EP0331961B1 (en) | Method for protein synthesis using CKS fusion proteins | |
| Kodaira et al. | The dnaX gene encodes the DNA polymerase III holoenzyme τ subunit, precursor of the γ subunit, the dnaZ gene product | |
| HU195534B (en) | Process for preparing polypeptide by bacterial way, by using tryptophan promoter operator | |
| ES8205263A1 (en) | Recombinant deoxyribonucleid acid which codes for plasminogen activator and method of making plasminogen activator protein therefrom. | |
| NO167673B (en) | PROCEDURE FOR PREPARING A POLYPEPTIDE. | |
| CA1210347A (en) | Microbial hybrid promotors | |
| CA1174617A (en) | Protein synthesis | |
| Collins | [20] Escherichia coli plasmids packageable in Vitro in λ bacteriophage particles | |
| JPH10500565A (en) | Expression plasmid controlled by osmB promoter | |
| US4649119A (en) | Cloning systems for corynebacterium | |
| IE52417B1 (en) | Dna sequences encoding various allelic forms of mature thaumatin,recombinant plasmids comprising said dnas and a process for their preparation,bacterial cultures comprising said recombinant plasmids,and method for producing mature thaumatin | |
| CA1193207A (en) | Structural genes encoding the various allelic maturation forms of preprothaumatin, recombinant cloning vehicles, comprising said structural genes and expression thereof in transformed microbial host cells | |
| EP0321940B1 (en) | An improved method for the preparation of natural human growth hormone in pure form | |
| Stuitje et al. | The nucleotide sequence surrounding the replication origin of the cop3 mutant of the bacterio-cinogenic plasmid Clo DF13 | |
| ATE161049T1 (en) | METHOD AND NUCLEIC ACID SEQUENCES FOR EXPRESSING THE CELLULOSE SYNTHASE OPERON | |
| Carter et al. | traY and traI are required for oriT-dependent enhanced recombination between lac-containing plasmids and lambda plac5 | |
| IE53607B1 (en) | Expression vectors | |
| US4631257A (en) | Stable high copy number plasmids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |