EP0390785A1

EP0390785A1 - Synthetic gene

Info

Publication number: EP0390785A1
Application number: EP19880908730
Authority: EP
Inventors: Richard Mark Edwards
Original assignee: British Bio Technology Ltd
Current assignee: Vernalis R&D Ltd
Priority date: 1987-10-08
Filing date: 1988-10-07
Publication date: 1990-10-10
Also published as: FI901763A0; GB8723661D0; JPH03501923A; DK88290D0; WO1989003422A1; DK88290A

Abstract

ADN synthétique codant la streptavidine, comprenant la séquence (I) et comportant des sites de restriction utiles à des intervalles fréquents, afin de faciliter la mutagénèse de cassettes de régions sélectionnées. Sont également inclus des sites de restriction latéraux afin de simplifier l'incorporation du gène dans n'importe quel système d'expression voulu.Synthetic DNA encoding streptavidin, comprising the sequence (I) and comprising restriction sites useful at frequent intervals, in order to facilitate the mutagenesis of cassettes of selected regions. Lateral restriction sites are also included to simplify incorporation of the gene into any desired expression system.

Description

SYNTHETIC GENE

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) .

In order to facilitate the dissection of the structure/function relationships of streptavidin, its incorporation into expression vectors and the production of novel chimeric proteins containing streptavidin functionality an improved novel synthetic gene for the streptavidin produced by Streptomyces avidinii is sought.

It is by no means easy to predict the design of an improved streptavidin gene, since the factors that determine the expressibility of a given DNA sequence are still poorly understood. Furthermore, the utility of the gene in various applications will be influenced by such considerations as codon usage and restriction sites. 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.

When synthesising and assembling genes, problems have been encountered when there are inverted or direct repeats greater than eight bases long in the genetic sequence. In addition, areas of unbalanced base composition such as G/C or A/T rich regions or polypurine/polypyrimidine tracts have been found to lead to inefficient expression. The present invention seeks to overcome or at least alleviate these difficulties.

According to a first aspect of the invention, there is provided DNA coding for streptavidin and having restriction sites for the following enzymes:

EcoRI, NdeI, PstI, SalI, SacI, SnaBI, BspMI , KpnI, FspI, PflMI, DraI , BalI, BstXI, HpaI, NsiI, SpeI, NheI, BstEII, XbaI, BamHI and HindIII

According to a second aspect of the invention, there is provided DNA including the following sequence:

ATG GAC CCG TCC AAG GAC TCC AAA GCT CAG GTT TCT GCA GCC GAA GCT GGT ATC ACT GGC ACC TGG TAT AAC CAA CTG GGG TCG ACT TTC ATT GTG ACC GCT GGT GCG GAC GGA GCT CTG ACT GGC ACC TAC GAA TCT GCG GTT GGT AAC GCA GAA TCC CGC TAC GTA CTG ACT GGC CGT TAT GAC TCT GCA CCT GCC ACC GAT GGC TCT GGT ACC GCT CTG GGC TGG ACT GTG GCT TGG AAA AAC AAC TAT CGT AAT GCG CAC AGC GCC ACT ACG TGG TCT GGC CAA TAC GTT GGC GGT GCT GAG GCT CGT ATC AAC ACT CAG TGG CTG TTA ACA TCC GGC ACT ACC GAA GCG AAT GCA TGG AAA TCG ACA CTA GTA GGT CAT GAC ACC TTT ACC AAA GTT AAG CCT TCT GCT GCT AGC ATT GAT GCT GCC AAG AAA GCA GGC GTA AAC AAC GGT AAC CCT CTA GAC GCT GTT CAG CAA TAA A synthetic streptavidin gene as described above incorporates useful restriction sites at frequent intervals to facilitate the cassette mutagenesis of selected regions. Also included are flanking restriction sites to simplify the incorporation of the gene into any desired expression system.

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.

Certain 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.

According to a third aspect of the invention, there is provided 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. Alternatively or in addition, 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. According to a fourth aspect of the invention, there is provided 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, the process 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.

Preferred embodiments and examples of the invention will now be described. In the following description, reference is made to a number of drawings, in which:

Figure 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; and

Figure 4 shows a summary of an assembly procedure used.

EXAMPLE

CONSTRUCTION OP THE GENE

The des ired gene s equence was d iv ided into 2 4 oligodeoxyribonucleotides (oligomers) as depicted in Figure 3. 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.

All the oligomers with the exception of the 5' terminal oligomers BB214 and BB237 were then kinased to provide them with a 5' phosphate as required for the ligation step. Complementary oligomers were then annealed and the 12 pairs of oligomers ligated together by T4 DNA ligase as depicted in Figure 4. The ligation products were separated on a 2% low gelling temperature (LGT) gel and the band corresponding to the 501/501 bp streptavidin gene duplex was cut out and extracted from the gel. The purified fragment was ligated to EcoRI/HindIII cut DNA of the plasmid vector pUC18. 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.

METHODS

All the techniques of genetic manipulation used in the manufacture of this gene are well known to those skilled in the art of genetic engineering. A description of most of the techniques can be found in the laboratory manual entitled Molecular Cloning by T. Maniatis, E.F. Fritsch and J. Sambrook published by Cold Spring Harbor Laboratory, Box 100, New York.

Additional and modified methodologies are detailed below.

1) Oligonucleotide synthesis

The 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)).

2) Purification of Oligonucleotides

The oligonucleotides were de-protected and removed from the CPG support by incubation in concentrated NH₃. 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.

For gel purification 10 absorbance units of the crude oligonucleotide were dried down and resuspended in 15 mcl of marker dye (90% de-ionised formamide, 10mM tris, 10 mM borate, ImM EDTA, 0.1% bromophenol blue). The samples were heated at 90° for 1 minute and then loaded onto a 1.2 mm thick denaturing polyacrylamide gel with 1.6 mm wide slots. The gel was prepared from a stock of 15% acrylamide, 0.6% bisacrylamide and 7M urea in 1 X TBE and was polymerised with 0.1% ammonium persulphate and 0.025% TEMED. The gel was pre-run for 1 hr. The samples were run at 1500 V for 4-5 hr. 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

250 pmole of oligomer was dried down and resuspended in 20 mcl kinase buffer (70 mM Tris pH 7.6, 10 mM MgCl₂, 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.

4 ) Annealing

8 mcl of each oligomer was mixed, heated to 90° and then slow cooled to room temperature over a period of an hour.

5) Ligation

5 mcl of each annealed pair of oligomers were mixed and 10 X ligase buffer added to give a final ligase reaction mixture (50 mM Tris pH 7.5, 10 mM MgCl₂, 20 mM dithiothreitol, 1 mM ATP. T4 DNA ligase was added at a rate of 100 u per 50 mcl reaction and ligation carried out at 15° for 4 hr.

6) Agarose gel electrophoresis

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.

8) Cloning of fragment

0.5 meg of pUC18 DNA (eg ATCC 37253) was prepared by cleavage with 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.

20 ng of cut vector DNA was then ligated to various quantities of streptavidin DNA ranging from 2 to 20 ng for 4 hr using the ligation buffer described above. The ligation products were used to transform competent HW87 as has been described. Ampicillin resistant transformants were selected on L-agar plates containing 100 meg ml^-1 ampicillin.

9) Isolation of plasmid DNA

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).

10) Dideoxy sequencing

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).

11) Transformation

Transformation was accompl ished using standard procedures . The strain used as a recipient in the cloning was HW87 which has the following genotype:

araD139 (ara-leu) del7697 (lacIPOZY) del74 galU galK hsdR rpsL srl recA56

Any other standard cloning recipient such as HB101 would be adequate.

Claims

1. DNA coding for streptavidin and having restriction sites for the following enzymes:

EcoRI, NdeI. PstI. SalI. SacI. SnaBI, BspMI, KpnI. FSPI. PflMI, DraIII, BalI. BstXI, HpaI, NsiI. SoeI. NheI, BstEII, XbaI. BamHI and HindIII

2. DNA including the following sequence:

ATG GAC CCG TCC AAG GAC TCC AAA GCT CAG GTT TCT GCA GCC GAA GCT GGT ATC ACT GGC ACC TGG TAT AAC CAA CTG GGG TCG ACT TTC ATT GTG ACC GCT GGT GCG GAC GGA GCT CTG ACT GGC ACC TAC GAA. TCT GCG GTT GGT AAC GCA GAA TCC CGC TAC GTA CTG ACT GGC CGT TAT GAC TCT GCA CCT GCC ACC GAT GGC TCT GGT ACC GCT CTG GGC TGG ACT GTG GCT TGG AAA AAC AAC TAT CGT AAT GCG GAC AGC GCC ACT ACG TGG TCT GGC CAA TAC GTT GGC GGT GCT GAG GCT CGT ATC AAC ACT CAG TGG CTG TTA ACA TCC GGC ACT ACC GAA GCG AAT GCA TGG AAA TCG ACA CTA GTA GGT CAT GAC ACC TTT ACC AAA GTT AAG CCT TCT GCT GCT AGC ATT GAT GCT GCC AAG AAA GCA GGC GTA AAC AAC GGT AAC CCT CTA GAC GCT GTT CAG CAA TAA

3. A genetic construct comprising DNA as claimed in claim 1 or 2, or a fragment thereof.

4. A construct as claimed in claim 3, wherein the fragment comprises at least 10 nucleotides.

5. A construct as claimed in claim 3, wherein the fragment comprises at least 20 nucleotides.

6. A construct as claimed in claim 3, wherein the fragment comprises at least 30 nucleotides.

7. A construct as claimed in claim 3, wherein the fragment comprises at least 40 nucleotides.

8. A construct as claimed in claim 3, wherein the fragment comprises at least 50 nucleotides.

9. A construct as claimed in claim 3, which is a vector.

10. A genetic construct as claimed in any one of claims 3 to 9 which is or embodies 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.

11. A process for the preparation of DNA as claimed in claim 1 or 2 or a genetic construct in accordance with claim 3, the process comprising coupling successive nucleotides and/or ligating appropriate oligomers.

12. DNA substantially as herein described with reference to Figure 2.