The dna fragment that encodes a protein gelonin, and method thereof, vector, ensuring the expression of the protein gelonin, the recombinant strain of e. coli producing protein gelonin

 

(57) Abstract:

The invention relates to biotechnology. The obtained synthetic gene that encodes a protein gelonin installed nucleotide sequence of SEQ ID NI, presented in the description. The method of obtaining includes a step of selecting the sequence of the DNA fragment, the stage of synthesis, which involves separating the DNA fragment into two overlapping set of single-stranded fragments of oligonucleotides. Synthesized chemically oligonucleotides selected from the group consisting of Btgel 1, Btgel 2 and gel 1 gel 39. Then they are cleaned, to phosphorylate the 5'-end. Annealing of the strands of the oligonucleotides together with the formation of duplex DNA leads to the formation of the synthetic gene. Obtained recombinant E. coli strain JM 105 transformed by the expression vector, which provides the expression of the protein gelonin. The vector contains a DNA fragment with the sequence SEQ ID NI, legirovannye in EcoRI/Hind III cleaved vector PKK 223-3. This recombinant technology allows to obtain reproductive rich source gelonin, which immunotoxic agent for the treatment of tumors. 4 S. p. f-crystals, 5 Il.

In General terms, the present invention relates to gelonin immunotoxin and SS="ptx2">

One of the reasons of interest at the present time to cytotoxic agents is their potential application in the field of specific nesenevich tumor cells. Plant toxin gelonin attracted such attention. Gelonin is a glycoprotein (MW. approximately 29-30000 KD) isolated from the seeds of Gelonium multiforum. Gelonin belongs to the class of potential plant toxins, inactivating ribosomes.

Other members of this class ribosome inactivating plant toxins are chain abrin, ricin and medecine. Gelonin like abrina and ricin, inhibits protein synthesis, damaging the 60S subunit of ribosomes mammals. Although A chain of ricin (RTA) is widely used in immunotoxin, gelonin seems more stable with respect to chemical and physical treatments than RTA (Barbieri et al, Cahcer Surv. 1:489-520 (1982). Moreover, he gelonin is not associated with the cells and therefore is non-toxic (except high concentrations) and with him to work safely in the laboratory. Inactivation of ribosomes reversible, apparently, does not include co-factors and occurs with efficiency, which involves the enzymatic mechanism of action gelonin.

Gelonin and the ricin is and. Gelonin in 10-1000 times more active in the inhibition of protein synthesis, rather than a chain of ricin A. Peptides such as ricin and abrina consist of two chains: A chain, which is a toxic element, and a B chain, which functions as communicating with the cell. Unlike ricin and abrin gelonin consists of one chain, and as it has no chain to bind to the cells, by itself it is relatively nontoxic to intact cells (Stirpe, et.al J. Biol. Chem. 255:6947-6953/1980). Mammalian cells, apparently unable to bind and/or internalize the native molecule gelonin. Conjugates gelonin with milenamimi for tumor monoclonal antibodies, such as monoclonal antibody ZME directed to an antigen that is present on some tumor cells such as melanoma cells, provide as a particular way of linking gelonin with cell and method of internalization of the complex gelonin-antibody. One of the advantages of using gelonin toxin compared with such toxins, as A chain of ricin is of low toxicity towards normal tissues compared with the toxicity of the A chain of ricin. Gelonin, coupled with a monoclonal antibody directed to anti-tumour antigen, t is ately use gelonin as a cytotoxic agent, chemically bound with monoclonal antibodies or peptide hormone cell milenamimi ligands. However, chemical modification gelonin and cell miseriya fragments can reduce micheneau efficiency and cytotoxic potential of gelonin. Moreover, natural sources gelonin are in various conditions in the collection and growth of plants, which may affect the cytotoxic activity gelonin. The ability to produce synthetic helaney toxin, chemically or by using recombinant technology, provides a rich, replicable source of the toxin. Therefore, it is highly desirable to obtain a synthetic gene gelonin and to develop methods of obtaining synthetic gene using recombinant technology.

The present invention provides the nucleotide sequence for a synthetic gene gelonin and how to obtain it. The DNA sequence of the synthetic gene gelonin presented in Sequence ID No. 1 (SEQID No. 1). The present invention also proposed the expression vectors containing such DNA sequences, and cells transformed with such vectors.

In accordance with one in the coding gelonin. First construct a fragment of the double DNA encoding the primary amino acid sequence, specific for purified gelasinospora protein. Then this DNA fragment is manipulated to facilitate synthesis, cloning and expression or biochemical manipulation of the genome.

Next, design, synthesize, purify and 5'-phosphorylate a set of synthetic oligonucleotides which are able to bind together with the formation of the desired gene. Then these oligonucleotides were annealed and are ligated together to obtain the intact gene. This synthetic gene are ligated together with a suitable cloning vector, and then determine the nucleotide sequence of the cloned gene. Then conduct a site-directed mutagenesis to correct any undesired mutations cloned gene, and this gene subcloning into a suitable expression vector. The expression vector carrying the synthetic helaney gene is inserted into a suitable host expression. After this, conditions for host expression support those who provide production gelasinospora gene product, a protein gelonin isolated and purified from cells expressing the gene.

In sommets nucleotides and fragments and derivatives thereof, encoding the protein gelonin or polypeptide that inhibits cellular protein synthesis, but does not bind the cell-surface receptors, and this nucleotide sequence has the formula sequence ID No. 1. Proposed the expression vector containing the synthetic DNA gelonin, and host cells containing and expressing a synthetic gene gelonin.

In accordance with another variant of the present invention is proposed immunotoxin containing antibody conjugated with plant protein toxin - gelonida.

For the purposes of the present invention, the term "fragment" is defined as any part of sequence ID No. 1, which can produce a protein that inhibits the synthesis of cellular protein, but which does not bind to the surface of the receptor. "Derivative" is defined as containing a substitution of one or more of the individual nucleic acids, so it's the same protein or polypeptide.

The present invention takes into account and addressed to the above mentioned essential necessary and sufficient satisfaction of this need and its various options. The specialists of this field will be ochevidnaya provides the following description together with the accompanying drawings. Although these descriptions are detailed to ensure adequacy and with the aim of better understanding, they should not limit the patent, the purpose of which is to say the invention regardless of how later someone will be able to disguise the different variations or additions of further improvements.

In order to make more understandable the details of the way (which the above features, advantages and objectives of the invention, and everything else that will become apparent hereinafter, are achieved), a more particular description of the invention, briefly summarized above, you can lead with reference to his some of the options that are illustrated by the attached drawings. These pictures are part of description. Note, however, that the appended drawings illustrate only preferred variants of the invention, and therefore they should not be construed as limiting its scope. The present invention may admit to other equally effective equivalent ways.

Fig. 1 depicts a schematic illustration of the method of chemical synthesis of oligonucleotides.

Fig. 2 depicts the process of solid-phase Assembly of a gene.

Fig. 3 is a schematic of degranulation in the Assembly of the gene gelonin.

Fig. 5 depicts the oligonucleotide sequence used to build gene gelonin.

Recent developments in molecular biology have enabled the cloning, expression and genetic engineering of many genes encoding proteins that represent values in Biomedicine and agriculture. Recent notable achievement in this area is the ability to design and produce synthetic genes. Synthetic genes may encode proteins of known amino acid sequences, as well as new proteins, which do not exist in nature. This is particularly useful in the case of proteins, representing therapeutic value, such as gelonin. It is possible to design, synthesize, clone and Express a gene on the basis of the amino acid sequence of the protein. Designing, sequencing, cloning and expressiona also possible, even if no specific information natural gene, for example, if a gene still has not been cloned. Moreover, the synthesis of gene facilitates the construction of the different gene products having properties that are not found in natural proteins. For example,ezerovo and cloned in the vector, which can lead to the production of large quantities of protein in the cells of microbial hosts.

One of the advantages of synthetic genes associated with the diversity of the genetic code. Most amino acids can be encoded by more than one "codon of three bases within a gene. Different organisms tend to use different codons for protein (S. Aota et. al., Nucleic Acids Res, v. 16, Supplement, p. r315-r391 (1987). In other words, the codons that are preferred in some organisms differ from those preferred in other organisms. Believe that this phenomenon is due to differences in the relative abundance of specific ioacceptor transport PHK molecules for a given amino acid. It is possible to construct synthetic genes to enable the preferred codons for a given expression system, even if the gene product is derived from "heterologous" of their host organisms, which use a different set of preferred codons for some amino acids. It was shown that synthetic genes encoding proteins of mammals, give significantly more protein product in a microbial host, if selected codons in the gene construct correspond to those on the s) intended for introduction into expressing host Escherichia coli. For expression in the system of another host gene gelonin easy to construct anew (this can be done by any expert in this field) to include codons that are optimal for another master.

Another advantage of synthetic genes, comply with the diversity of the genetic code, is that the DNA sequence encoding a gene of interest can be modified (without changing the encoded amino acid sequence) so that it contains the maximum number of sites of recognition of the unique restriction enzymes in this gene. A plurality of unambiguous restriction sites in the gene facilitates biochemical manipulation of this gene. For example, the ability to cut a segment of the gene and replace it with another DNA sequence (which is ensured by close of unique restriction sites) facilitates the introduction of new genetic information in the gene using recombinant DNA techniques. Convenient manipulation offered by such approaches include the introduction of mutations in certain parts of the gene, fix evolution is the dominant proteins, which combine the functional domains of the individual proteins.

The design of the synthetic gene may also include non-coding "flanking DNA sequences that can facilitate cloning and gene expression. For example, the sequence recognition restriction endonuclease, you can include in the flanking sites of the gene to ensure specific ligation of the gene in target vector cloning. In addition, the genetic signals can be embedded in a synthetic gene which is used for monitoring gene expression in vivo.

In addition, the newly designed gene may facilitate introduction into a specific location in vivo gene product in some tissues or organs, which can improve therapeutic effect of gene product. For example, it facilitates the directed introduction of a therapeutic agent to a particular cell type by introducing into the corresponding gene sequence which encodes a polypeptide domain which specifically binds to a receptor on the surface nesenevich cells.

The design of the synthetic gene can also be included in such consideration, which are associated with the chemical synthesis co it is important to avoid weak binding efficacy, associated with the subsequent addition of many G or C residues in the chain. In addition, it is desirable to avoid DNA sequences that contain minitalia secondary structure (structure "studs") or intermolecular complementarity, which can interfere with proper Assembly of the gene during annealing component oligonucleotides. These goals can be achieved by choosing an alternative codons in the gene construct.

The method of Assembly of genes includes the following stages. First allocate the nucleotide sequence corresponding to the two strands of the target coding of the site, providing a good pairing of complementary bases between the two threads. Then add any desired flanking sequences. For example, you can add flanking sequences to enable the recognition sites of restriction endonuclease adjacent to the coding sequence. Then the gene is divided into overlapping sets of single-stranded fragments. Single-stranded fragments are chemically synthesized in a device for automated DNA synthesis. The degree of complementary overlap between consecutive oligonucleotides together with a synthetic genome is a matter of choice, n is a mini-gene, preferably purified by electrophoresis on polyacrylamide gel or by using high-performance liquid chromatography. Then purified oligonucleotides 5'-phosphorylate under the action of polynucleotide kinase and adenosine-5'-tryptophane. Then thread annealed together, either in a separate mixture in blocks 3-10 overlapping oligonucleotides, or stepwise by adding oligonucleotides on a solid phase carrier. The ends of the assembled gene provide restriction sites used for cloning the gene. The assembled gene is usually first clone in the single-stranded M13 vector for convenient DNA sequencing. If necessary, the correct mutation oligonucleotide directed mutagenesis.

The desired characteristics of the synthetic gene (optimal use of the codon, the existence of a unique restriction sites, with the exception of the secondary structure, and so on ) can be identified using any of a number of commercial programs analysis of DNA sequences for microcomputers.

Two recent developments, both of which are used in the method of the present invention, provide a more rapid and efficient synthesis of genes and create new opportunities for the design of Protea Fig. 1, is a technology for fast efficient synthesis of large numbers of oligonucleotides. This technology allows you to get all synthetic DNA, required for Assembly of the gene in one day. In accordance with Fig. 1 treated with nucleoside glass with controlled pore size is placed in a separate cell synthesis, consisting of a Teflon cylinder with a porous ends, which allows fluid to flow through the stack of cells. The simultaneous addition of A, G, C or T for DNA chains attached to the CPG and remaining inside the cells, is due to the sequential flow of reagents through the column due to phosphoramidite method (L. I. McBride and M. N. Caruthers, Tetrahedron Lett. , 24, 245-248 (1983)). After each of the cycles of chemical reactions of the cell are sorted by different columns to ensure the synthesis of different nucleotide sequences within each cell. Shows the serial position of a single cell (shaded) in four cycles, which should result in the addition of AGCT to the growing DNA chain contained in it.

The second technological development, which facilitates the synthesis is the creation of tools for sequential joining of synthetic oligonucleotides on TV is a set of overlapping complementary oligonucleotides. Assembly begins with the binding of one end of the nucleotide with a solid-phase carrier. 5'-phosphorylated oligonucleotides added sequentially (with a molar excess) to a related media thread. At each stage of Assembly of a gene unrelated DNA wash before conducting the following reaction annealing. After Assembly, all treated with DNA ligase for ligating single-stranded breaks, then the gene is separated from the media by cleavage at a unique restriction site contained in a linked with the carrier of the oligonucleotide. Released DNA are ligated into a suitable vector for sequencing and expression.

Many variations exist for the host vector when the expression of the synthetic gene, which leads to the production of the encoded protein. They are discussed in detail in the Methods Enzymology, Yol 152, 1987, Academic Press. In short, a special expression vectors available for inclusion in bacterial, fungal, animal or plant hosts. The most commonly used Escherichia coli for the expression of "alien" genes. Another popular host for expression are the yeast Saccharomyces cerevisial. As mentioned earlier, if interest cloned gene synthesized him to increase the level of expression of the engineered gene. Most expression vectors contain genetic regulatory elements, located adjacent to the cloning site, which provides high levels of gene expression. Inducible promoters expression vectors are typically derived from bacteria (e.g., TAS, thr) or viruses (e.g., lambda, SV 40). The elements of the signal sequence is sometimes included in the vector to control the transport of the gene product from the host cells. Elements of signal sequences can facilitate cleaning and reduce proteolytic degradation. In the case of synthetic genes any target genetic control element may be included to collect the DNA duplex. Some expression vectors containing the coding sequence, following immediately after the cloning site so that the insert in the frame of the foreign coding sequence may be provided inside of the collected sequence in the case of synthetic genes. The production of the gene as a fusion protein provides a number of advantages, including increased expression, higher stability, rapid affinity purification using associated with a native ligand that binds to an additional polypeptide to the mi, possessing on their surface receptors for additional polypeptide components). For the further development of therapies based gelonin can be used in genetic engineering, including this last feature.

Cloning and expression of DNA sequences encoding plant toxin gelonin, you can use a wide range of vectors. They include, for example, vectors, consisting of segments of chromosomal, achromosome and synthetic DNA sequences such as various known derivatives of SV 40, plasmids from E. coli including col E1, pCR1, pBR322, pMB9 and their derivatives, a wide range of plasmids owners (such as RP4, phage DNA), for example, various derivatives of phage lambda, (e.g., NM 989), and other DNA phages (e.g., M13 and filamentous single-stranded DNA phages, plasmids, yeast, such as 2mu plasmid or its derivatives, and the vectors derived from combinations of plasmids and DNA phages, such as plasmids that have been modified to use the DNA of a phage or other sequences controlling the expression.

For each specific cloning or expression vector can be selected various sites for insertion of DNA sequences present is haunted. Well-known different ways of embedding the DNA sequences in these sites for obtaining recombinant DNA molecules. They include, for example, capacity dG-dC or dA-dT direct ligation, synthetic linkers, reactions repair associated with exonuclease and polymerase with posleduyushim legirovaniem, or elongation of DNA strands DNA polymerase and the appropriate a single-stranded template with subsequent legirovaniem. Of course, you should be aware that the cloning vectors or expression suitable for the present invention should not be sites of restriction endonucleases to activate the selected DNA fragment. Instead, the carrier can be attached to the movie in other ways.

For expression of the DNA sequence of the present invention, these DNA sequences are operatively linked to one or more of the sequences that control expression in the expression vector. Such operative linking, which can be done before or after the selected DNA sequence is included in the cloning vector, provides the expression control sequences and promotion of the expression of the integrated DNA sequence.

For obraznyh sequences regulating expression. Such appropriate regulatory expression sequence include, for example, early and late promoter, SV 40, the lac system, the trp system, the TAC or TRC system, major operator and promoter of phage lambda, the control sections of the fd protein shell, the promoter for 3-phosphoglycerate or other glycolytic promoters, the promoters of acid phosphatase, e.g., Pho5, the promoters of yeast factors alpha-mating and other sequences known to control the expression of genes in prokaryotic and eukaryotic cells or their viruses, and various combinations thereof. In mammalian cells it is also possible to amplify fragments of the expression, by binding gene with coded dehydropeptidase and conducting selection on ovary cells Chinese hamster.

The vector or carrier of expression, the specific sites selected for embedding the selected DNA fragment and a sequence controlling the expression used in the present invention, determine the various factors. For example, these factors include the number of sites corresponding to a particular restriction enzyme, size of the subject expression of the protein characteristics such expressions, including idny specialists. The choice of the vector, expression control sequences and incorporating site-specific sequence of the protein inhibitor of phospholipase determine the ratio of these factors, and not always this choice is equally effective in a particular case.

Recombinant DNA molecule containing the target gene, operatively linked to a sequence controlling the expression can then be used to transform a wide range of relevant hosts in order to ensure the expression of these hosts (transformants) of the gene or its fragment and get the polypeptide or part thereof, which encodes a hybrid DNA.

To obtain antigens and DNA sequences of the present invention can be used with a wide host range. These hosts include, for example, bacteria, such as E. coli, Bacillus and Streptomyces, fungi such as yeasts, animals, such as CHO cells, and plant cells in tissue culture. Choosing the right host is determined by a number of factors that are known to experts. They include, for example, compatibility with the chosen vector, the toxicity of co-products, easy selection of the target peptide, expression characteristics, bio-safety Oh recombinant DNA molecule or polypeptide. Instead, you should consider these factors, given that not all hosts can be equally effective for the expression of specific rekombinantnoi DNA molecules.

As shown previously, it should be considered that DNA sequences that build on the chosen site media cloning or expression may contain nucleotides that are not part of the real gene encoding the target polypeptide, or may include only a fragment of the gene of this protein. It is only necessary that regardless of the DNA sequence, transformed host was produced protein gelonin or polypeptide with almost the same functional activities that gelonin. For example, DNA sequences of the present invention can be drained in the same reading frame in the expression vector, the DNA sequence that encodes at least one prokaryotic or eukaryotic protein, or a DNA sequence that encodes at least one eukaryotic or prokaryotic signal sequence or a combination thereof. Such structures may be used as the expression of the desired DNA sequence, to improve OCOM embodiment, the DNA sequence may include ATG start codon, alone or together with other codons, merged directly with the sequence encoding the first amino acid of the target polypeptide. Such designs provide, for example, metioning or other peptidergic polypeptides, that is part of the invention. Then the N-terminal methionine or peptide can be split intra - or extra-cellular various known methods or polypeptide can be used together with methionine or other fused and attached to it, in the compositions and methods of the present invention.

Examples

Synthesis and Assembly of gene gelonin

Example 1. The binding of 5'-biotinylated oligonucleotides with latex beads coated with streptavidin.

A sample of 0.2 ml DYNABEADS M280 (Denal Corp.) placed in Eppendorf vials with a capacity of 1.5 ml Vials incubated on a magnetic plate (Advanced maghetics, Ync) for several minutes in order to cause precipitation of the latex microspheres on the one hand, and then the fluid is drained. The beads washed twice with 0.2 ml origaudio buffer (composition given below) at room temperature, then re-suspended in 0.2 ml origaudio buffer.

To a suspension of beads was added 1 nmol 5'-biotinylated is of buffer and again suspended in 0.2 ml origaudio buffer. Spectrophotometric analysis of the unbound oligonucleotide in the leaching shows that approximately 300 pmoles of the oligonucleotide is associated with balls.

Example 2. The cycle of annealing/rinse repeat to complement each subsequent nucleotide.

Before use in the Assembly of the gene, the oligonucleotides were purified by electrophoresis on polyacrylamide gel and enzyme, 5'-phosphorylate using T4 polynucleotide.

To 150 polam associated with the carrier of the oligonucleotide add 750 pmoles overlapping complementary oligonucleotide, and annealing are in 0.10 ml of 50 mm nutrifaster buffer, pH 7.5, 1M NaCl (buffer annealing) for 5 minutes at 45oC, then the mixture is cooled to room temperature for 7 minutes. Then the beads are washed twice with 0.2 ml of the same buffer at room temperature. This cycle is repeated until then, until you add the last of the oligonucleotide in the Assembly.

Example 3. Ligation product and release from the carrier by digestion with restriction enzyme.

After Assembly is complete, the beads are washed again and suspended in 0.04 ml ligase buffer. After the addition of 0.005 ml DNA ligase (New England Biolabs, with high specific is antiroot 0.04 ml restriction digest buffer. After adding 10 units of restriction endonuclease EcoR1, the mixture is incubated at 37oC for 90 minutes. The fluid is drained, and the liberated DNA is precipitated with ethanol and re-suspended in 0.01 ml ligase buffer.

Example 4. The Assembly of the gene gelonin.

This gene are collected from both directions, as shown in Fig. 3 and Fig. 4. Oligonucleotide sequences are shown in Fig. 5. The Assembly of the 5'-end of the gene (approximately 500 bp of the N-terminal coding section) start with associated with the media Btgel1, and oligonucleotides were added in the following order (each spend cycle annealing/flushing): gel39, gel1, gel38, gel2, gel37, gel3, gel36, gel4, gel35, gel5, gel34, gel6, gel33, gel7, gel32, gel8, gel31, gel9, gel30, gel10, gel29, gel11, gel28, gel12, gel27.

The Assembly of the 3'-end of the gene (approximately 300 bp C-terminal coding section) start with associated with the media Btgel2, and oligonucleotides were added in the following order: gel20, gel19, gel18, gel22, gel17, gel23, gel16, gel24, gel15, gel25, gel14, gel26, gel13.

In accordance with Fig. 3 5'-end of the gene (the N-terminal coding section) released from the carrier, restriction digesting their endonuclease EcoR1 and 3'-end of the gene (C-terminal encoding section) released from the carrier, digesting with restriction endonuclease H is the nucleotide gel27 and gel13, annealed together, and then are ligated to obtain the intact gene.

Example 5. Cloning of the synthetic gene gelonin.

Complete DNA product are ligated M13mp19RF DNA, which was digested EcoR1 and Hind111, by the standard method described in Molecular Cloning: a Laboratory Handbook, EF. Sambrook et. al, 1989.

Example 6. Sequencing synthetic gene gelonin.

The sequence of the synthetic gene into M13mp19 confirm dideoxy-sequencing. In the cloned synthetic gene detected two mutations, both with 5'-end (N-terminal coding section).

Example 7. Site-directed mutagenesis to correct mutations in the cloned synthetic gene.

Spend oligonucleotide-directed mutagenesis to correct two mutations within a gene gelonin by the way, supplied with in vitro by a set of reagents mutagenesis (Amersham Corp.).

Example 8. Subclavian synthetic gene into the expression vector.

Synthetic helaney gene otscheplaut from M13mp19 vector under the action of EcoR1 and Hind111 and containing the gene fragment purified by agarose gel electrophoresis, and then are ligated into EcoR1/Hind111-cleaved expression vector pKK223-3 (Pharmacia).

the synthetic gene gelonin, cloned into pKK223-3, is grown, induce IPTG and spend lysis to obtain the crude extract. The extract analyzed by electrophoresis on acrylamide gel (along with the control extract derived from the host cells containing the expression vector without inserts). The Western blot testing and functional analysis gelonin also performed to confirm that the protein is expressed and active.

In conclusion, we should say that the present invention disclosed here its variants are well adapted to carry out the objectives and obtain the specified results. In the method and apparatus may be amended, without going beyond the spirit and scope of the present invention. Note that changes are possible and that each element or stage, following in the following claims should be considered as equivalent elements or stages to implement practically the same goals and almost in the same ways. The invention should be considered broadly, no matter in what form will be used for his ideas. Therefore, the present invention is well adapted for solving problems and getting results and prealloc gelonin, with nucleotide sequence (see sequence N1 in the section "Tables").

2. Vector providing the expression of the protein gelonin containing the DNA fragment under item 1, legirovannye with EcoRI/HindIII cleaved vector PKK 223-3.

3. Recombinant Escherichia coli strain JM 105 transformed by the expression vector for p. 2 producing protein gelonin.

4. A method of obtaining a synthetic gene encoding a protein gelonin providing stage of the selection sequence of the DNA fragment containing the sequence encoding gelonin, and this sequence contains a triplet codon of three bases for each amino acid in the sequence gelonin and triplet chosen from the group of triplets coding for each amino acid, to maximize expression, manipulation or stability of the DNA fragment into the expression vector by synthesizing the specified portion, the synthesis of the DNA fragment, and the synthesis involves separating the DNA fragment into two overlapping set of single-stranded fragments, oligonucleotides, and fragments synthesized chemically and oligonucleotides selected from the group consisting of Btgel 1, Btgel 2 and gel 1 gel 39, cleaning these oligonucleotides, FOSFA is of duplex DNA which leads to the formation of a synthetic gene encoding gelonin.

 

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