DD263994A1 - PROCESS FOR PREPARING OLIGODESOXYRIBONUCLEOTIDES FOR SPLENOPENTINE EXPRESSION - Google Patents

Abstract

The invention relates to a process for the preparation of oligodeoxyribonucleotides of the general formula 1 which are insertable and clonable in prokaryotic and eukaryotic vectors and which code the expression of splenopentines in both prokaryotic and eukaryotic systems. 1 Oligodeoxyribonucleotide Duplexes: According to the invention, the novel double-stranded oligodeoxyribonucleotides with splenopentin information are obtained by carrier-assisted phosphotriester synthesis of the coding and non-coding oligodeoxyribonucleotide strand and their subsequent hybridization to prokaryotic and eukaryotic vectors, directly inserting, cloning and within fusion protein information in prokaryotic and eukaryotic systems expressive splenopentines. Splenopentin are immunomodulatory.

Description

wherein the restriction enzyme recognition regions with 5 'resp. 3'-protruding cohesive or blunt ends for the in the reading direction (arrow) initial restriction step optionally contain 1 to 2 additional deoxyribonucleotide pairings for adjustment of the reading frame, the Met code with

is inserted as coding of the later BrCN cleavage sites of the fusion proteins, the splenopentine encodings - including the codons which can also be used as a result of code degeneration - are represented by the following sequences:

Splenopentin (SP-5) Arg-Lys-Glu-Val-Tyr

Prokaryote Expression (E. coli) 5'-CGT-AAA-GAA-GtA-TAC-S '

3'-GCA-TTT-CTT-CAT-ATg-B 'Eukaryote Expression (Yeast) 5'-AGA-AAG-GAA-GTT-TAc-S'

3'-TCT-TTC-CTt-CAA-ATG-B 'Pro ² splenopentin (Pro? -SP-5) Arg-Pro-Glu-Val-Tyr

Prokaryote expression (E. coli)

3'-GCA-GGC-CTt-CAT-ATG-B 'Eukaryote Expression (Yeast) 5'-AGA-CCA-GAA-GTT-TAc-S'

3'-TCT-GGT-CTT-CAA-ATg-S 'pentyne (I) Arg-Pro-Gln-Val-Tyr

Prokaryote Expression (E. coli) B'-CGT-CCG-CAG-GTA-TAC-S '

3'-GCA-GGC-GTC-CAT-ATG-B '

Eukaryote Expression (Yeast) B'-AGA-CCA-CAA-GTT-TAC-S '

3'-TCT-GGT-GTT CAA ATG-B '

Pentyne (II) Arg-Pro-Gln-Pro-Tyr

Prokaryote expression (E. coli) B'-CGT-CCG-CAG-CCG-TAC-S '

3'-AGA CCA CAA CCA TAC-S '

Eukaryote expression (yeast) B'-AGA-CCA-CAA-CCA-TAC-S '

3'-TCT-GGT-GTTGGT ATG B '

Pentin (III) Glu-Pro-Gln-Va! -Tyr

Prokaryote expression (E. coli) S'-GAA-CCA-CAG-GTA-TAC-S '

3'-CCT-GGC-CAT-GTC-ATG-B '

Eukaryote expression (yeast) B'-GAA-CCA-CAA-GTT-TAC-S '

3'-CCT GGT GTT CAA ATG-B '

and, as stop codes, find 1-2 condons of the following three triplets:

3'-ATTATCACT-B '

be prepared by supported Phosphotriestersynthesen and then hybridized.

Field of application of the invention

The invention relates to a seduction for the preparation of oligodeoxyribonucleotides of the general formula 1, which are insertable and clonable in prokaryotic and eukaryotic vectors and encode the expression of splenopentines.

1 Oligodeeoxyribonucleotide dojppelets:

Re8trict: Lonecut Mat-Spleinopentin- I etop- - Coding Reetriktione- cut

Pfail ; Lens direction ---- Splenopentines are immunomodulatory in their effect.

Characteristic of the known technical solutions

Splenopentin-type peptides are so far only as partial sequencing of the corresponding precursor protein. Splenin (T. Audhya et Oil.,

Biochemistry 20 (1981) 6195) or as structural analogues. Splenopeniin-5 (SP-5) (Diezel, W., et al., Biomed., B'ochim, Acta 43 (1984 K9), has been synthesized peptide-synthesized in a number of structural analogues so far only the thymopentin-5 gene sequence has been described in any of the C. C. Russells et al., DE 3102 721A1, Jan. 28, 1981.) A direct chemical synthesis of splenopentienes, as well as the genes themselves, are not yet known for the production of nucleic acid sequences Various methods are known, such as the phosphodiester, the Phosphotriester- and the Phosphora nidilverfahren (HG <. Gassen and A. Lang, Chemical and enzymatic synthesis of gene fragments - a laboratory manual, Vrlag Chemie, Weinheim 1982).

Aim of the invention

The object of the invention is the chemical synthesis of double-stranded oligodeoxyribonucleotides which are capable of expression for splenopentines due to their specific strand, code and reading frame design in prokaryotic and eukaryotic vectors, and in prokaryotic and eukaryotic host systems.

Explanation of the essence of the invention

The object of the invention is a method by which corresponding oligodeoxyribonucieotide strands can be produced which code the expression of splenopentines as hybrid fourth double strands.

According to the invention the Oiigodesoxyribonucleotidstränge of the general formula 1 with:

Restriction step:

Met-coding:

Restrlktionsenzymerkennungsbereiche with 5'- or 3'-protruding cohesive or blunt ends, for Jen in the reading direction initial restriction step, where appropriate, involving 1 to 2 additional deoxyribonucleotide pairs for adjustment of the reading frame. 5'-ATG-3 'as coding of the later 3'-TAC-5' 8rCN cleavage site of the fusion proteins

Splenopsntin of the general formula A-B-C-D-Tyr

peptide

Splenopontin (Sp-5) bad Arg-Lys-Glu-Val-Tyr Lys Glu Pro ² splenopentin bad Per Glu (Pro ² -SP-5) Pentyne (l) bad Per Gin Pentyne (II) bad Per Gin Pentyne (III) Glu Per Gin Splenopentin encodings: Splenopentin (Sp-5)

VaI VaI

VaI Pro VaI

Prokaryoton Expression (E. coli) Eukaryote Expression (Yeast) Pro ² -splenopentin (Pro ² -Sp-5)

Prokaryote Expression (E. coli) Eukaryote Expression (Yeast) Pentin (l)

5'-CGT-AAa-GAA-GTA-TAC-S '3' · GCA-TTT-CTT-CAT-ATG-B '5'-AGA-AAG-GAA-GTT-TAc-S' 3'-TCT- TTC-CTT-CAa-ATG-B 'Arg-Pro-Glu-Val-Tyr

5'-CGT-CCG-GAA-GTa-TAC-S '3'-GCA-GGC-CTT-CAT-ATg-B' 5'-AGA-CCA-GAA-GtT-TAC-S '3-TCT-GGT -CTT-CAa-ATG-B 'Arg-Pro-Gln-Val-Tyr

Prokaryote expression (E. coli) Eukaryote expression (yeast) Pentin (II)

5'-CGT-CCG-CAG-GTA-TAc-S '3'-GCA-GGC-GTC-CAt-ATG-B' B'-AGA-CCA-CAA-GTT-TAC-S '3'-TCT GGt-GTT-CAA-ATG-S 'Arg-Pro-Gln-Pro-Tyr

Prokaryote Expression (E. coli) Eukaryote Expression (Yeast) Pentin (III)

5'-CGT-CCG-CAG-CCG-TAc-S '3'-GCA-GGc-GTC-GGC-ATG-B' B'-AGA-CCA-CAA-CCA-TAC-S '3'-TCT- GGT-GTT-GGt-ATG-B 'Glu-Pro-Gln-Val-Tyr

Prokaryote expression (E. coli)

Eukaryotic expression (yeast)

5'-GAA CCG CAG GTA TAC-S '

3'-CTT-GGC-GTC-CAT-ATG-B '

5'-GAA CCA CAA GTT TAC-S '

3'-CTT-GGT-GTt-CAA-ATG-B 'Stop Encoding: 5'-TAA TAG TGA-3' Use of 3'-ATT-ATC-ACT-B '1-2 Triplets

and all splenopentin encodings including codons useful as a result of code degeneracy in place of the above triplets prepared by a supported phosphotriester synthesis. Hybridizations to insertable, cloning and expression splenopentin duplexes are performed by conventional techniques (T. Maniatis et al., Molecular cloning - a laboratory manual, Cold Spring Harbor, 1982, and B. Levin in gene expression Vol. Wiley lntercience 1980).

The selection of splenopentin information triplets was based on the expression strategies of the underlying pro- and eukaryotic systems (J.L. Bennetzen et al., Biol. Chem. 257 [1982] 3026) and the requirements for additional restriction recognition for restriction mapping of the insertions and cions ,

The chemically synthesized splenopentine genes are due to their cohesive or smooth restriction sections immediately insertion, cloning and expression capable in prokaryotic and eukaryotic systems. Depending on the specificity of the respective vectors, 0-2 additional deoxyribonucleotide pairings ensure the alignment of the reading frames in the transition of the initial restriction steps to the start of release codes.

The splenopentin information, the actual splenopentine gene, precedes met start information, ensuring the subsequent splitting off of the splenopentin information from the fusion proteins, and the splenopentine's downstream stop signals ensuring proper termination.

The release of splenopentin information from the fusion proteins released from prokaryotic and eukaryotic systems produces the immunomodulatory splenopentin.

The direct chemical synthesis of these splenopentynes, which are directly insertable, clonable and expressible in prokaryotic and eukaryotic systems, is like the. Splenopentingene itself - new.

embodiments

Synthesis of splenopentin (Sp-5) gene 28mer: noncoding strand 26mer: coding strand

EcoRI Met Arg Lye GIu VaI Tyr stop CIaI

1) 28mer 5'-AATTC ATG CGT AAA GAA GTA TAC TAA AT -3 '

2) 26mer 3 ¹ - G TAC GCA TTT CTT CAT ATG ATT TAGC-5 '

L Accil Sn a I

To realize the given gene structure, a 26- and a 28-mer oligodeoxyribonucleotide were synthesized. For synthesis, a modified Phosphotriesterverfahren on solid phase in the 5'-3 'direction was selected.

The chain construction takes place in single steps in a manually operated closed apparatus.

The corresponding 2'-deoxyribonucleotides were protected by N-acylation in the base part by a known method and dimethoxytrityliert in sugar in δ'-Ροελίοη. The phosphorylation of the 3'-OH group was carried out with p-Chlorphenylphosphorditriazolid to the corresponding monotriazolide and with 2-cyanoethanol to fully protected deoxyribonucleoside S'-monophosphorsäuretriester. This was followed by acid cleavage of the 4,4'-dimethoxytrityl protecting group from the 5'-end. These compounds were used after chromatography on silica gel.

The final building block was a base-protected and 3'-acetylated nucleoside.

According to Scheme 1, a stepwise construction was carried out at a cycle time of 20-22 min.

The carrier used was a 12% Teflon-grafted polystyrene which was provided with an aminomethyl group. The amino group was determined by means of picric acid titration. In our case, it was 170μητιοΙ-NH ₂ / g carrier. The spacer was succinic acid. The loading of the carrier was carried out with 5'-succinyl-nucleoside-3'-p-chlorophenyl-2-cyanoethylphosphorsäuretriester in the presence of dicyclohexylcarbodiimide (DCC). It was a nucleotide loading in the case of 1) of 76μπιοΙ dAp / g (at 20mg carrier use = 1.5μπιοΙ) and at 2) of ΘΟμιποΙ dCp / g (at a carrier use of 1,2μιτιοΙ) achieved. The starting point was 20mg derivatized vehicle. It was treated with a 10-fold excess of nucleotide component

It-if

in absolute pyridine, a 30-fold excess condensing agent (triisopropylbenzenesulfonyl chloride TIPS) and a slight excess of nucleophilic catalyst 1-methylimidazole (MeIm) worked (based on the nucleotide load of the carrier). After the transfer of the carrier into the reactor to block the still free NH ₂ groups was a "zero condensation" with TIPS / 1-methylimidazole (10:30) in 10min.

Scheme 1

surgery

reagent

Time

1. drying the carrier

2. Cleavage of the CNEt protecting group

3. Washing

4. Condensation

5. Washing

pyridine

0.1 m tetramethylguanidine in abs. pyridine

Section. pyridine

Coupling mixture b) in abs. pyridine

pyridine

a)

1-2 min

a)

12-15 min

a)

a) The washing operations are completed when the relative conductivity of the final value of the abs. Pyridine has reached.

b) The coupling mixture consists of a mixture of 10 equiv of the dried nucleotide component, added 30 fiquiv. TIPS and 90 equiv of 1-methylimidazole in abs. Pyridine (0.1-0.15 molar) based on the initial loading of the carrier.

After completion of synthesis and washing with ether, the support was transferred to a screw top sealed tube, with 30% of NH ₄ OH + 10% pyridine and heated overnight at 5O ⁰ C. After filtration and washing with pyridine, it was carefully concentrated to dryness. The amount of crude product was determined by UV spectroscopy. In the 28mer it was 295 ODE and in the 26mer 240 O. D E. Part of this crude product (of the total amount) was purified on a 20% denatured polyacrylamide gel (20 x 40 x 0.3 cm). The markers used were XC, BPB and corresponding reference substances. The longest UV-active bands were cut, crushed and water added. After about 5 h at 5O ⁰ C incubation was centrifuged from) polyacrylamide and the clear supernatant was desalted on DEAE-Sephacel. After washing with water, 0.1 M TEAB, the corresponding oligodeoxyribonucleotide was eluted from the column with 1 M TEAB solution and concentrated carefully with the addition of pyridine. The purified end product was measured by UV spectroscopy. As proof, an autoradiography of the two single strands was carried out after 5'-labeling with subsequent sequencing on CCS paper (A.Rosenthal et al., Nucleic Acids Res., 13 (1985) 1173.) Furthermore, the restriction sites were ligated by ligation of the hybridized strands over the EcoRI and cial sites and corresponding resections are checked with additional use of the Accl sections.

The hybridization itself was according to Maniatis et al. (T. Maniatis) et al. Molecular cloning - a laboratory manual. Cold Spring Harbor, 1982).

The yields for the 28mer after the polyacrylamide purification were 23.9% = 94.8% / stage and for the 26mer 16.7% = 93.1% / stage.

Abbreviations: xylene cyanol (XC)

Bromophenol blue (BPB)

Claims (1)

Process for the preparation of oligodeoxyribonucleotides for splenopentine expression, characterized,. in that the oligodeoxyribonucleotide strands of the general formula 1 Restriktlonssohnltt Met Splenopentln-Oodierung atop- Eeetriktionssohnitt