WO1989003422A1 - Synthetic gene - Google Patents
Synthetic gene Download PDFInfo
- Publication number
- WO1989003422A1 WO1989003422A1 PCT/GB1988/000831 GB8800831W WO8903422A1 WO 1989003422 A1 WO1989003422 A1 WO 1989003422A1 GB 8800831 W GB8800831 W GB 8800831W WO 8903422 A1 WO8903422 A1 WO 8903422A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gct
- dna
- ggc
- acc
- aac
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
Definitions
- This invention relates to synthetic genes coding for streptavidin.
- Streptavidin is a 60 kD protein isolated from Streptomyces avidinii that binds extremely tightly to the vitamin biotin with a dissociation constant of about 10 -15 M. Streptavidin is composed of four identical subunits of 15 kD and binds 4 mole biotin per mole of protein. It is structurally related to the protein avidin, a biotin binding protein isolated from chicken egg whites.
- Streptavidin has particular utility in that it can be readily conjugated to a range of other proteins such as horseradish peroxidase and alkaline phosphatase. This has allowed the development of sensitive detection systems for biotinylated oligonucleotides, antibodies and other binding proteins with application in the analysis of DNA, RNA, protein and carbohydrates.
- the natural gene for streptavidin has been described and was found to encode a protein of 183 amino acids which included a typical leader sequence of 24 amino acids. The N-terminus of the mature protein was found to correspond to residue 25 of the deduced sequence.
- the amino acid sequence of mature streptavidin deduced from the gene sequence was used as the basis of the design for the synthetic streptavidin genes of the present invention and is shown in Figure 1.
- the binding of streptavidin to biotin was described in 1963 by Chaiet et al. (Agents Chemother. 3, 28-32).
- the cloning of. the natural streptavidin gene and its sequence together with the deduced streptavidin amino acid sequence is taught by Argarana et al. (Nucleic Acids Res. 14 1871-1882 (1986)).
- the production of streptavidin-like polypeptides is disclosed in EP-A-0198015 (Biogen NV) .
- the present invention relates to a synthetic streptavidin gene which is distinct from the natural streptavidin gene and has advantages in the ease with which it can be modified due to the presence of useful restriction sites.
- DNA including the following sequence:
- Codons are those that are favoured by E. coli but it is expected that the DNA would be suitable for expression in other organisms including yeast and mammalian cells.
- DNA sequences in accordance with the invention encode the entire mature streptavidin protein together with the required initiator methionine residue but lack the leader sequence present in the natural gene. It is envisaged that the leader sequence appropriate to the expression system, of choice would be added to the synthetic gene as required or omitted to allow for intracellular expression of the gene.
- a genetic construct comprising DNA according to the first or second aspect or a fragment thereof.
- the fragment may comprise at least 10, 20, 30, 40 or 50 nucleotides.
- a genetic construct in accordance with the third aspect may be a vector, such as a plasmid, cosmid or phage.
- a genetic construct in accordance with the third aspect may be a chimeric gene composing all or a fragment of DNA according to the first or second aspect fused to any other sequence of DNA so as to result in a sequence capable of encoding a hybrid protein possessing biotin binding activity.
- a process for the preparation of DNA in accordance with the first or second aspect or a genetic construct in accordance with the third aspect comprising coupling successive nucleotides and/or ligating appropriate oligomers.
- the invention also relates to other nucleic acid (including RNA) either corresponding to or complementary to DNA in accordance with the first or second aspects.
- FIG 1 shows the amino acid sequence of streptavidin
- Figure 2 shows a sequence of a streptavidin synthetic gene in accordance with the invention incorporating a summary of restriction sites
- Figure 3 shows a sequence of a synthetic streptavidin gene in accordance with the invention divided into oligonucleotides
- Figure 4 shows a summary of an assembly procedure used.
- oligomers oligodeoxyribonucleotides
- the division was such as to provide 7 base cohesive ends after annealing complementary pairs of oligomers.
- the end points of the oligomers were chosen to minimise the potential for inappropriate ligation of oligomers at the assembly stage.
- the oligomers were synthesised by automated solid phase phosphoramidite chemistry. Following de-blocking and removal from the controlled pore glass support the oligomers were purified on denaturing polyacrylamide gels, further purified by ethanol precipitation and finally dissolved in water prior to estimation of their concentration.
- the ligated product was transformed into HW87 and plated on L-agar plates containing 100 meg ml -1 ampicillin. Colonies containing potential clones were then grown up in L-broth containing ampicillin at 100 meg ml -1 and plasmid DNA isolated. Positive clones were identified by direct dideoxy sequence analysis of the plasmid DNA using the 17 base universal primer, a reverse sequencing primer complementary to the opposite strand on the other side of the polylinker and some of the oligomers employed in the assembly of the gene that served as internal primers. One streptavidin clone was subsequently re-sequenced on both strands to confirm that no mutations were present.
- oligonucleotides were synthesised by automated phosphoramidite chemistry using cyanoethyl phosphoramidtes. The methodology is now widely used and has been described (Beaucage, S.L. and Caruthers, M.H. Tetrahedron Letters 24, 245 (1981)).
- the oligonucleotides were de-protected and removed from the CPG support by incubation in concentrated NH 3 .
- concentrated NH 3 typically, 50 mg of CPG carrying 1 micromole of oligonueleotide was de-protected by incubation for 5 hr at 70° in 600 mcl of concentrated NH3.
- the supernatant was transferred to a fresh tube and the oligomer precipitated with 3 volumes of ethanol. Following centrifugation the pellet was dried and resuspended in 1 ml of water. The concentration of crude oligomer was then determined by measuring the absorbance at 260 nm.
- the bands were visualised by UV shadowing and those corresponding to the full length product cut out and transferred to micro-testubes.
- the oligomers were eluted from the gel slice by soaking in AGEB (0.5 M ammonium acetate, 0.01 M magnesium acetate and 0.1 % SDS) overnight.
- the AGEB buffer was then transferred to fresh tubes and the oligomer precipitated with three volumes of ethanol at -70° for 15 min.
- the precipitate was collected by centrifugation in an Eppendorf microfuge for 10 min, the pellet washed in 80 % ethanol, the purified oligomer dried, redissolved in 1 ml of water and finally filtered through a 0.45 micron micro-filter.
- the concentration of purified product was measured by determining its absorbance at 260 nm. 3) Kinasing of oligomers
- oligomer 250 pmole of oligomer was dried down and resuspended in 20 mcl kinase buffer (70 mM Tris pH 7.6, 10 mM MgCl 2 , 1 mM ATP, 0.2 mM spermidine, 0.5 mM dithiothreitol). 10 u of T4 polynucleotide kinase was added and the mixture incubated at 37° for 30 min. The kinase was then inactivated by heating at 85° for 15 min.
- Ligation products were separated using 2% low gelling temperature agarose gels in 1 X TBE buffer (0.094 M Tris pH 8.3, 0.089 M boric acid, 0.25 mM EDTA) containing 0.5 meg ml -1 ethidium bromide. 7) Isolation of ligation product
- the band corresponding to the expected streptavidin gene ligation product was identified by reference to size markers under long wave UV illumination. The band was cut out of the gel and the DNA extracted as follows.
- the volume of the gel slice was estimated from its weight and then melted by incubation at 65° for 10 min. The volume of the slice was then made up to 400 mcl with TE (10 mM Tris pH 8.0, 1 mM EDTA) and Na acetate added to a final concentration of 0.3 M. 10 meg of yeast tRNA was also added as a carrier. The DNA was then subjected to three rounds of extraction with equal volumes of TE equilibrated phenol followed by three extractions with ether that had been saturated with water. The DNA was precipitated with 2 volumes of ethanol, centrifuged for 10 min in a microfuge, the pellet washed in 70 % ethanol and finally dried down. The DNA was taken up in 20 mcl of TE and 2 mcl run on a 2 % agarose gel to estimate the recovery of DNA.
- pUC18 DNA eg ATCC 37253
- HindIII and BamHI as advised by the suppliers.
- the digested DNA was run on an 0.8 % LGT gel and the vector band purified as described above.
- Plasmid DNA was prepared from the colonies containing potential streptavidin clones essentially as described (Ish-Horowicz, D., Burke, J.F. Nucleic Acids Research 9 2989-2998 (1981).
- the protocol used was essentially as has been described (Biggin, M.D., Gibson, T.J., Hong, G.F. P.N.A.S. 80 3963-3965 (1983)).
- the method was modified to allow sequencing on plasmid DNA as described (Guo, L-H., Wu, R. Nucleic Acids Research 11 5521-5540 (1983).
- araD139 (ara-leu) del7697 (lacIPOZY) del74 galU galK hsdR rpsL srl recA56
- Any other standard cloning recipient such as HB101 would be adequate.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NO90901588A NO901588L (en) | 1987-10-08 | 1990-04-06 | SYNTHETIC GEN. |
FI901763A FI901763A0 (en) | 1987-10-08 | 1990-04-06 | SYNTHETIC GEN. |
DK088290A DK88290A (en) | 1987-10-08 | 1990-04-06 | SYNTHETIC GEN |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB878723661A GB8723661D0 (en) | 1987-10-08 | 1987-10-08 | Synthetic gene |
GB8723661 | 1987-10-08 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1989003422A1 true WO1989003422A1 (en) | 1989-04-20 |
Family
ID=10624999
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1988/000831 WO1989003422A1 (en) | 1987-10-08 | 1988-10-07 | Synthetic gene |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0390785A1 (en) |
JP (1) | JPH03501923A (en) |
DK (1) | DK88290A (en) |
FI (1) | FI901763A0 (en) |
GB (1) | GB8723661D0 (en) |
WO (1) | WO1989003422A1 (en) |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2212160B (en) * | 1987-11-13 | 1991-12-11 | British Bio Technology | Dna sequences encoding the bspmi recognition site. |
WO1993009144A1 (en) * | 1991-10-28 | 1993-05-13 | Boehringer Mannheim Gmbh | Recombinant core-streptavidin |
WO1994000992A1 (en) * | 1992-07-10 | 1994-01-20 | Pioneer Hi-Bred International, Inc. | Avidin and homologues as larvicides against insect pests |
WO1997011183A1 (en) * | 1995-04-11 | 1997-03-27 | Trustees Of Boston University | Streptavidin mutants |
WO1997011186A1 (en) * | 1995-09-23 | 1997-03-27 | Boehringer Mannheim Gmbh | Process for producing natriuretic peptides via streptavidine fusion proteins |
WO1997018314A1 (en) * | 1995-11-16 | 1997-05-22 | Boehringer Mannheim Gmbh | Process for the preparation of peptides by way of streptavidin fusion proteins |
US5973116A (en) * | 1993-01-15 | 1999-10-26 | Imperial Cancer Research Technology Limited | Compounds for targeting |
US6156493A (en) * | 1995-02-09 | 2000-12-05 | University Of Washington | Modified-affinity streptavidin |
WO2002072817A1 (en) * | 2001-03-12 | 2002-09-19 | Japan Tobacco Inc. | Novel protein, gene encoding the same and method of using the same |
US6492492B1 (en) | 1998-04-03 | 2002-12-10 | University Of Washington | Circularly permuted biotin binding proteins |
WO2005014650A2 (en) | 2003-06-16 | 2005-02-17 | Celltech R & D, Inc. | Antibodies specific for sclerostin and methods for increasing bone mineralization |
US6972350B1 (en) | 1998-07-15 | 2005-12-06 | The Horticulture And Food Research Institute Of New Zealand | Pest-resistant plants comprising a construct encoding a vacuole targeting sequence and avidin or streptavidin |
EP2275816A2 (en) | 2006-03-22 | 2011-01-19 | Viral Logic Systems Technology Corp. | Methods for identifying polypeptide targets and uses thereof for treating immunological diseases |
EP2322933A1 (en) | 2001-08-29 | 2011-05-18 | Pacific Northwest Research Institute | Diagnosis of carcinomas |
EP2338906A1 (en) | 2003-06-16 | 2011-06-29 | UCB Manufacturing, Inc. | Compostion and methods for increasing bone mineralization |
US8377448B2 (en) | 2006-05-15 | 2013-02-19 | The Board Of Trustees Of The Leland Standford Junior University | CD47 related compositions and methods for treating immunological diseases and disorders |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1983004053A1 (en) * | 1982-05-06 | 1983-11-24 | Applied Molecular Genetics, Inc. | The manufacture and expression of large structural genes |
WO1986002077A1 (en) * | 1984-10-02 | 1986-04-10 | Meade Harry M | Production of streptavidin-like polypeptides |
WO1987005026A1 (en) * | 1986-02-24 | 1987-08-27 | The Trustees Of Columbia University In The City Of | Dna encoding streptavidin, streptavidin produced therefrom, fused polypeptides which include amino acid sequences present in streptavidin and uses thereof |
EP0171024B1 (en) * | 1984-08-10 | 1991-09-11 | Hoechst Aktiengesellschaft | Genetic engineering process for the production of hirudins, and agents for carrying out said process |
-
1987
- 1987-10-08 GB GB878723661A patent/GB8723661D0/en active Pending
-
1988
- 1988-10-07 EP EP19880908730 patent/EP0390785A1/en not_active Ceased
- 1988-10-07 WO PCT/GB1988/000831 patent/WO1989003422A1/en not_active Application Discontinuation
- 1988-10-07 JP JP50816688A patent/JPH03501923A/en active Pending
-
1990
- 1990-04-06 DK DK088290A patent/DK88290A/en not_active Application Discontinuation
- 1990-04-06 FI FI901763A patent/FI901763A0/en not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1983004053A1 (en) * | 1982-05-06 | 1983-11-24 | Applied Molecular Genetics, Inc. | The manufacture and expression of large structural genes |
EP0171024B1 (en) * | 1984-08-10 | 1991-09-11 | Hoechst Aktiengesellschaft | Genetic engineering process for the production of hirudins, and agents for carrying out said process |
WO1986002077A1 (en) * | 1984-10-02 | 1986-04-10 | Meade Harry M | Production of streptavidin-like polypeptides |
WO1987005026A1 (en) * | 1986-02-24 | 1987-08-27 | The Trustees Of Columbia University In The City Of | Dna encoding streptavidin, streptavidin produced therefrom, fused polypeptides which include amino acid sequences present in streptavidin and uses thereof |
Cited By (33)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2212160B (en) * | 1987-11-13 | 1991-12-11 | British Bio Technology | Dna sequences encoding the bspmi recognition site. |
WO1993009144A1 (en) * | 1991-10-28 | 1993-05-13 | Boehringer Mannheim Gmbh | Recombinant core-streptavidin |
AU659240B2 (en) * | 1991-10-28 | 1995-05-11 | Boehringer Mannheim Gmbh | Recombinant core-streptavidin |
WO1994000992A1 (en) * | 1992-07-10 | 1994-01-20 | Pioneer Hi-Bred International, Inc. | Avidin and homologues as larvicides against insect pests |
US5973116A (en) * | 1993-01-15 | 1999-10-26 | Imperial Cancer Research Technology Limited | Compounds for targeting |
US6165750A (en) * | 1995-02-09 | 2000-12-26 | University Of Washington | Modified-affinity streptavidin |
US6156493A (en) * | 1995-02-09 | 2000-12-05 | University Of Washington | Modified-affinity streptavidin |
WO1997011183A1 (en) * | 1995-04-11 | 1997-03-27 | Trustees Of Boston University | Streptavidin mutants |
US6022951A (en) * | 1995-04-11 | 2000-02-08 | Univ Boston | Streptavidin mutants |
US6207390B1 (en) * | 1995-04-11 | 2001-03-27 | The Trustees Of Boston University | Methods for the use of reduced affinity streptavidin |
WO1997011186A1 (en) * | 1995-09-23 | 1997-03-27 | Boehringer Mannheim Gmbh | Process for producing natriuretic peptides via streptavidine fusion proteins |
WO1997018314A1 (en) * | 1995-11-16 | 1997-05-22 | Boehringer Mannheim Gmbh | Process for the preparation of peptides by way of streptavidin fusion proteins |
US6136564A (en) * | 1995-11-16 | 2000-10-24 | Roche Diagnostics Gmbh | Process for the production of peptides by way of streptavidin fusion proteins |
US6492492B1 (en) | 1998-04-03 | 2002-12-10 | University Of Washington | Circularly permuted biotin binding proteins |
US6972350B1 (en) | 1998-07-15 | 2005-12-06 | The Horticulture And Food Research Institute Of New Zealand | Pest-resistant plants comprising a construct encoding a vacuole targeting sequence and avidin or streptavidin |
EP1369476A4 (en) * | 2001-03-12 | 2004-07-28 | Japan Tobacco Inc | A novel protein, a gene encoding therefor and a method of using the same |
EP1369476A1 (en) * | 2001-03-12 | 2003-12-10 | Japan Tobacco Inc. | A novel protein, a gene encoding therefor and a method of using the same |
WO2002072817A1 (en) * | 2001-03-12 | 2002-09-19 | Japan Tobacco Inc. | Novel protein, gene encoding the same and method of using the same |
EP1865059A1 (en) * | 2001-03-12 | 2007-12-12 | Japan Tobacco, Inc. | A novel protein, a gene encoding therefor and a method of using the same |
KR100890839B1 (en) * | 2001-03-12 | 2009-03-27 | 니뽄 다바코 산교 가부시키가이샤 | A novel protein, a genes encoding therefor and a method of using the same |
US7713531B2 (en) | 2001-03-12 | 2010-05-11 | Japan Tobacco, Inc. | Protein, a gene encoding therefor and a method of using the same |
US7776333B2 (en) | 2001-03-12 | 2010-08-17 | Japan Tobacco Inc. | Protein, a genes encoding therefor and a method of using the same |
US7855282B2 (en) | 2001-03-12 | 2010-12-21 | Japan Tobacco Inc. | Protein, a gene encoding therefor and a method of using the same |
US7989610B2 (en) | 2001-03-12 | 2011-08-02 | Japan Tobacco Inc | Protein, a gene encoding therefor and a method of using the same |
EP2913673A1 (en) | 2001-08-29 | 2015-09-02 | Pacific Northwest Research Institute | Diagnosis of ovarian carcinoma |
EP2322933A1 (en) | 2001-08-29 | 2011-05-18 | Pacific Northwest Research Institute | Diagnosis of carcinomas |
EP2341071A1 (en) | 2003-06-16 | 2011-07-06 | UCB Manufacturing, Inc. | Compostion and methods for increasing bone mineralization |
EP2338906A1 (en) | 2003-06-16 | 2011-06-29 | UCB Manufacturing, Inc. | Compostion and methods for increasing bone mineralization |
WO2005014650A2 (en) | 2003-06-16 | 2005-02-17 | Celltech R & D, Inc. | Antibodies specific for sclerostin and methods for increasing bone mineralization |
EP2322931A2 (en) | 2006-03-22 | 2011-05-18 | Viral Logic Systems Technology Corp. | Methods for identifying polypeptide targets and uses thereof for treating immunological diseases |
EP2275816A2 (en) | 2006-03-22 | 2011-01-19 | Viral Logic Systems Technology Corp. | Methods for identifying polypeptide targets and uses thereof for treating immunological diseases |
US8293500B2 (en) | 2006-03-22 | 2012-10-23 | Viral Logic Systems Technology Corp. | Methods for identifying polypeptide targets and uses thereof for treating immunological diseases |
US8377448B2 (en) | 2006-05-15 | 2013-02-19 | The Board Of Trustees Of The Leland Standford Junior University | CD47 related compositions and methods for treating immunological diseases and disorders |
Also Published As
Publication number | Publication date |
---|---|
DK88290A (en) | 1990-06-08 |
GB8723661D0 (en) | 1987-11-11 |
FI901763A0 (en) | 1990-04-06 |
EP0390785A1 (en) | 1990-10-10 |
JPH03501923A (en) | 1991-05-09 |
DK88290D0 (en) | 1990-04-06 |
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