HU202580B - Process for producing reverse transchriptase of virus of pons sarcoma with recombinative dns-technic - Google Patents

Abstract

The present invention relates to a method for producing reverse transcriptase of a Rous sarcoma virus by recombinant DNA technology. The reverse gene transcriptase encoding the reverse transcriptase from plasmid pSRA2 derived from Rous sarcoma virus is ligated to a perfectly inducible lac promoter that can be propagated in an E. coli bacterium into plasmid pUC9, with E. coli transformations selected by the ligate, and repression and expression of a vital pol gene selected by selection. recombinants containing sequences of mRNA binding to the ribosome and translational initiator and terminator sequences, thereby transforming E. coli, bacterial cells, and purifying the extracted, raw reverse transcriptase, if desired. (Figure 3) EN 202 580 A Scope of the description: 6 pages, 3 drawings -1-

Description

The present invention relates to a method for the production of reverse transcriptase of Rous sarcoma virus by recombinant DNA technology.

Reverse transcriptase is an enzyme found in so-called retroviruses that contain a single-stranded RNA genome that infects eukaryotic cells. Reverse transcriptase is essential for the life cycle of such viruses. It is used to transcribe the viral single-stranded RNA genome into double-stranded DNA. The annealed double-stranded vfrus DNA is sequentially cleaved by the reverse transcriptase at the annealing site and the linearized virus genome can be incorporated into the host cell genome (transformation).

The reverse transcriptase enzyme catalyzes a primer- and template-dependent reaction: it produces the complement of single-stranded RNA DNA from the DNA, and the resulting RNA / DNA hydride becomes double-stranded with the same enzyme. In this process, in addition to synthesizing activity, it also contributes to the so-called RNase H activity of the enzyme, which, prior to the formation of double-stranded DNA, removes or hydrolyses the RNA component of the DNA / RNA hydride.

The reverse transcriptases isolated from different sources have different molecular weights, typically between 70,000 and 110,000 daltons. Other properties of the enzyme also depend on the source. For example, reverse transcriptase isolated from mice retroviruses has a monomeric structure and reverse transcriptase derived from avian retroviruses is a subunit with a larger beta subunit protease to a smaller alpha subunit and a third protein (p32). et al., J. Virol. 33, 264 (1980); Schwartz et al., Cell, 22, 853 (1983). Among them, βζ, αβ and its components have reverse transcriptase activity.

Reverse transcriptase is one of the key and at the same time one of the most expensive enzymes in molecular biological research. By using it, virtually every RNA sequence can be transcribed into a DNA strand (complementary, i.e., cDNA) which can then be cloned, amplified, sequenced, manipulated, etc. into a suitable vector. More recently, it has been used extensively to "fill" single-stranded DNA, and for DNA sequencing instead of DNA polymerase. The enzyme is expensive because commercially available reverse transcriptase enzyme preparations are prepared by the conventional method from virally infected cells. The virus is first isolated from the cells and the enzyme itself is extracted from the resulting preparation by further, lengthy and laborious purification steps. Producing a larger amount of enzyme in this way is very time consuming, labor intensive and therefore expensive. For example, the following reverse transcriptase preparations are commercially available:

Pharmacia Reverse Transcriptase, AMV 27-0921-01 200 E

Boehringer Reverse Transcriptase 109-118 1000 E

Amersham Reverse Transcriptase T. 26104 300 E (E = Unit)

Recently, reverse transcriptase has also been produced by recombination DNA techniques for scientific purposes. Tanese et al., Proc. Natl. Acad. Sci. U.S.A. 87, 4944 (1985)] and Kotewicz et al. (Gene, 35,249 (1985)) have cloned a minor central portion of the Moloney murine leukemia virus reverse transcriptase gene (pol gene) to produce a reverse transcriptase having an enzymatic activity in E. coli. After proper selection, the resulting product had satisfactory enzyme activity, but the authors did not prove that the synthesized DNA matched the template. According to M. L. Kotewicz, even worse results should be expected in the case of cloning of avian retroviruses, such as the avian myeloblastosis virus pol gene (AMV), primarily due to the subunit nature of the reverse transcriptase enzyme of this origin. Namely, during the synthesis of native AMV reverse transcriptase, the polymerase gene product must form a β-β dimer, which is then phosphorylated and cleaved at a specific site into one β-protein subunit, the so-called. α-subunit and p32 protein. The physiologically active reverse transcriptase is a dimer of α and β subunits. These are processes that are not catalyzed in E. coli.

In the process of the present invention, our aim was to produce high-quality recombinant DNA technology using standard quality enzymes that closely approximate the properties of native AMV reverse transcriptase, which is considered to be the best and commonly used.

Contrary to what can be expected in the literature, it has been found that cloning the reverse transcriptase gene from avian Rous sarcoma virus with an appropriately selected expression vector in E. coli provides an enzyme preparation that meets the above requirement.

In particular, we have discovered that the reverse transcriptase coding gene from Rous sarcoma virus [DeLorbe et al., J. Virol. 36, 50 (1980)] by cloning in a suitable vector for propagation in E. coli, the reverse transcriptase enzyme is produced in large quantities in the living bacterial cell, from which it can be isolated by conventional techniques and then purified by a relatively simple and inexpensive method.

Thus, the present invention provides a method for the production of a reverse transcriptase of Rous sarcoma virus by inserting a Rous sarcoma virus reverse transcriptase coding plasmid derived from plasmid pSRA2 into an E. coli bacterial, fully repressible, inducible ligand, lac promoter, E. coli is transformed by selection by selection of a recombinant containing repression, expression, sequence binding to the mRNA ribosome and sequence translational initiator and terminator by selection of a viral pol gene. crude reverse transcriptase enzyme was purified.

The genome of the Rous sarcoma virus is known at the sequence level from Schwartz et al., Cell, 32, 853 (1983). The reverse transcriptase enzyme gene is derived from pSR A2 plasmid carrying the double-stranded NDS copy (SR A2) of Schmidt Ruppin A Rous sarcoma virus (see DeLorbe et al., Supra). The structure of plasmid pSRA2 is illustrated in Figure 1.

HU 202 580 A

The most important requirements for a high copy number recombinant plasmid for use in the method of the invention are as follows:

- Have a strong promoter that

- be well inducible,

- be perfectly repressible, otherwise the host cell and eventually the cloning site will be destroyed during transformation

- put the pol herons into a proper translation phase.

Examples of such plasmids are the plasmids of the pUC series, e.g. pUC7, pUC8, pUC9, etc. (Villeia and Messing, Gene, 19259 (1982)). Plasmids containing the Ihc promoter:

(DeBoer et al., In Promoter Structure and Function Ed. LR. Rodrigez and M. J. Chamberlain, Praegar Publ. N. Y. 1982).

PL promoter vectors [Hedgpeth et al., Mol. Gene 163, 197, 1978].

Vectors containing the Trp E promoter [Tacan et al., Mol. Gene. GeneL 177, 427 (1980)].

Preferably, the present invention proceeds as follows: The commercially available high copy number plasmid pUC 9 (Figure 2) is opened in its polylinker with Pst I and Sal I, and the DNA between the Pst I and Xho I sites is inserted into this genes. section. With the resulting ligandE. coli strain HB101. Colonies carrying the desired recombinant plasmid are then selected as follows:

A small amount of colonies were lysed and the DNA was electrophoresed on an agarose gel according to Eckhardt (Maniatis et al., 1982, Laboratory Handbook, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.). The recombinant plasmid carrying the insert of about 2.8 kb (pMF 14) can be selected without difficulty.

The bacterial strain containing the desired recombinant is propagated to generate sufficient plasmids for mapping (see Maniatis et al., 1982). Each plasmid was cleaved with various restriction enzymes to verify that the desired construct was obtained [DcLorbe et al., J. Virol. 36.50 (1980)].

Based on the mapping performed, it can be seen that the pMF 14 construct carrying the viral pol gene (Figure 3) has all of the sequence fragments that provide for the regulated transcription of the cloned gene, binding to the mRNA ribosome, translation initiation and terminator sequences. The regulatory elements of the expression of the cloned gene - operator, promoter, Shine-Dalgamo sequence, translation initiation triplet - are derived from the E. coli lac operator and the components of plasmid pUC9, while the translation codon is provided by the pol gene itself. The N-terroinal end of the gene product was supplemented with a 25 amino acid peptide. Of these, 9 are amino acids from the vector, the rest are derived from a transcription of the sequence prior to the natural start of the pol protein. The sequence of the extra peptide is as follows: (See) Thr Met De Thr Pro Ser Leu Ala Gly Pro Arg Ala Pro Leu Asp Lys Phe Ile Gly Arg Thr Val Leu.

Plasmid pMF 14 was used to transform E. coli strain HB101. Plasmid-bearing cells were able to proliferate without difficulty in standard liquid and solid media such as LB, overlaid on agar plates prepared in the presence of ampicillin. The stability of the plasmid and thus of the cloned gene is ensured by always containing 0.5% glucose in addition to the required components in the medium. Maximum expression of the pol gene can be induced by IPTG. Cells carrying plasmid pMF 14 are then grown on a rich liquid medium containing 0.5% glucose, while glucose over-metabolites are depleted of the medium. This usually occurs at Eoo = 2.2 densities. Cells are harvested by centrifugation and resuspended in glucose-free medium (M9) containing 10% of the original volume of glucose-free other components required for E. coli strain HB101 (Maniatis et al., 1982). After incubation at 30 poc at 37 ° C, IPTG is added to 10 mM and incubated for a further 90 minutes. The biomass collected by centrifugation can be used immediately for enzyme preparation or stored in dry ice at -20 ° C until use.

This is followed by cell digestion and centrifugation, where the carefully aspirated supematant is the extract containing the desired enzyme. Purification of the extract from ion exchange resins and. dialysis to obtain a pure enzyme with enzyme activity determined under standard reaction conditions ((50 mM Tris, pH 8.3), 50 mM KCl, 6 mM MgCl ₂ , 5 mM dithiothreitol, 0.8 µg poly / rA.dTi _S and 0.5 mM pH] dTTP in 20 µl at 37 ° C for up to 20 stains] 5-20 units / 1 µΐ About 10-20,000 U can be obtained from 1 liter of bacterial culture.

The main advantages of the process according to the invention are as follows:

1. In practice, the most commonly used enzyme is avian virus (AMV), which is well known for producing the most perfect cDNA. Structurally, our bird cloned RSV reverse transcriptase is also close to this enzyme. Our recombinant enzyme can be produced in unlimited amounts in high concentrations (> 20 U / pl) and the cost of production is far below the cost of producing a conventional enzyme.

2. Kotewicz et al. 1985 and Thnese et al. 1985 The Moloney sarcoma virus (mouse) reverse transcriptase gene has been cloned, but only a portion thereof. In contrast, we have cloned the entire reverse transcriptase coding region.

3. Our enzyme preparation is stable. At least 85% of his activity was maintained within 6 months. In contrast, others have reported a high degree of lability of their product [M. J. Roth et al. al., J. Bioi. Chem. 260, 9326 (1985)].

4. Isolation of our recombinant enzyme does not require ionic detergent (eg quantitatively recovered without SDS). Denaturing agent adversely affects enzyme quality In contrast to our procedure, Kotewicz et al. [Gene 35, 249 (1985)] can mobilize a greater proportion of the enzyme using SDS.

5. The cloning of the RSV pol gene allows for a complete genetic engineering of the naturally-occurring αβ subunit. The latter has been shown to be the active form of the virus, which is the most accurate

The cDNA makes a copy of the virus or whatever

RNA template.

The following is a non-limiting example of the process of the present invention. However, for the sake of clarity, before the example is given, a summary of the figures and all the tools and methods used in the exemplary embodiment of the procedure are given.

FIGURE DESCRIPTION The aim was always to supplement the medium with freshly diluted ampicillin to a concentration of 50-100 pg / ml. In all cases where the reverse transcriptase gene was also present in the plasmid, glucose was added to a concentration of 0.5%.

M9 (so-called minimum medium):

Maniatis et al., 1982

Agar plates (1.5% Bacto agar) were stained with the above media.

Cultures were performed at 37 'C with vigorous shaking in the case of liquid media.

Figure: Schematic structure of the pSRA2 plasmid carrying the RSV genome, showing cleavage sites important for cloning. H3 = = Price ΠΙ. DeLorbe et al., J. Virol. 36, 50 (1980) and Grandgenett et al., J. Virol. 33,264 (1980) and Grandgenett et al., J. Virol. 33, 264 (1980)]

Figure 2: Schematic structure of pUC 9

Figure 3: Schematic structure of plasmid pMF 14 H3 = Hind III; Ri. = EcoRI.

Figure 4: Incubation of snRNA with recombinant reverse transcriptase 37 'C, 4 hours = RNA preparation incubated with 10 U reverse transcriptase = RNA preparation incubated without reverse transcriptase = RNA preparation without incubation.

Plasmid Preparation

Plasmid was isolated from 10-20 ml of culture according to the commonly used method (Maniatis et al., 1982).

For the isolation of larger amounts of plasmid (500-1000 ml culture), Norgard (Norgard, Anal. Biochem. 113, 34, (1981)) was used, whereby the supernatant obtained after centrifugation of the bacterial chromosomal DNA was purified by BioGel A. The 5M column was gel-filtered and the fractions containing the plasmid were centrifuged in a CsCl equilibrium density gradient.

gel electrophoresis

DNA samples were electrophoresed in Tris-borate 0.7-0.9% agarose gel (Maniatis et al., 1982).

Figure 5: Synthesized with various reverse transcriptases solutions: comparison of cDNA Lysing solution: l% Triton X-100 1 = 7.5 U recombinant reverse Drosophila polyA + mRNA 25 02% NP-40 transcriptase 1 mM EDTA 2 = 12.5 E Boehringer Reverse 2 mM DTT transcriptase 2 mM PMSF

mM Na phosphate, pH 8.0

3 = 7.5 U recombinant reverse transcriptase; poly A ⁺ rat liver hnRNA Materials 40 Buffer A: 10% glycerol 5% sucrose 0.2% NP-40 10 mM Na-phosphate, pH 8.0 1 mM DTT 0.5 mM EDTA T4 ligase and restriction endonucleases were found in the USSR. 0.1 mM PMSF from Union-produced and marketed preparations. 45 0.1 M NaCl The reagents and resins used are anilytically qualitative. Buffer B: 10% glycerol most of them are Sigma, Merck, Bio-Rad, Phar- 02% NP-40 macean, products of Reanal. 10 mM Na-phosphate, pH 8.0 50 1 mM DTT Methods 0.5 mM EDTA 0.1 mM PMSF DNA cleavage with restriction endonucleases for in vitro recombination, ligation, and bacterial transformation. Buffer C: 75 mM K-phosphate, pH 7.3 was performed according to commonly used methods 55 7 mM β-mercaptoethanol (Maniatis et al., 1982, cited above). 02% NP40 0.1 M EDTA Culture media and cultures 10% glycerol LB: Bacto trypton 10 g 60 Buffer D: 50% glycerol Bacto yeast extractum 5 g 2 mM DTT NaCl 10 g 1 mM EDTA H ₂ O to 1000 ml. 0.2% NP 40 200 mM K-phosphate, pH 12 When the plasmid-bearing cells were cultured 65

HU 202 580 A

Example 1

6 µg of pSRA2 DNA are taken and treated in succession with restriction enzymes Xma I, Xho I and Pst I. After digestion, agarose gel electrophoresis was performed to check the efficiency of the digestion. Μΐ pUC9 DNA was digested with PstI and SalI as described above.

Both DNAs are dissolved in 20-20 µl of ligation buffer after ethanol precipitation and three times 70% wash in ethanol (Maniatis et al., 1982). 20 ng of cut pUC9 and 1350 ng of cut pSRA2) DNA are mixed, supplemented with the required components (Maniatis et al., 1982), 10 units of ligase are added to the reaction mixture and kept overnight in an 8 ° C refrigerator.

An E. coli strain HB101 competent for calcium chloride methods (Maniatis et al., 1982) was transformed with the ligate mixture (5-10 μΐ) and plated on LB medium supplemented with 05% glucose and 20 μΐ / ml ampicillin. .

after incubation for 1 hour (37 'C), the grown colonies were analyzed for the size of the plasmid contained therein (Maniatis et al., 1982).

Bacterial strains containing the expected approximately 5.5 kb plasmid are selected. These cells were grown in liquid medium containing glucose and ampicillin and a plasmid was isolated (Maniatis et al., 1982). The plasmid was screened with restriction endonucleases (PstI, SalI, Bam HI, Hind HI) or combinations thereof (Maniatis et al., 1982) and compared to the original map (DeLorbe et al., 1980).

The selected clone is used for enzyme testing as follows:

2 μΐ of the overnight culture in LB medium supplemented with glucose and ampicillin was inoculated with 20 ml of fresh medium above and incubated with vigorous shaking at 37 DEG C. to a density of E1.0 = 1.0.

EPTG, freshly dissolved in water, was then added to the culture to an ImM concentration and incubated for an additional 60 minutes.

Cells harvested by transfection are treated with lysozyme (Maniatis et al., 1982) and lysed with spheroplasts (Materials and Methods 1). 10000 g of supematant is used directly for enzyme testing.

The controlled and reverse transcriptase producing clone is grown and induced at a preparative size and the product isolated from the culture for characterization of the enzyme.

Example 2

Preparative isolation of reverse transcriptase

E. coli HB 101 containing 800 ml of pMF 14 plasmid was grown in LB medium supplemented with glucose (0.5%) and ampicillin (100 pg / ml) to a density of 2.2. After centrifugation, the cells were resuspended in 50 ml glucose-free (ampicillin-containing) M9 medium and incubated with vigorous shaking for 30 minutes to 10 mM IPTG and incubated for an additional 15 hours. Cells collected by centrifugation were frozen in liquid air and stored until -20 'Con processing.

Each step of enzyme preparation is carried out at 4 'C.

Frozen cells were resuspended in 8 ml of 10% sucrose solution containing 10 mM Na phosphate, pH 8.0, and 2 ml of 20 mg / ml lysozyme solution (dissolved in the above buffer) was added. After 10 minutes, 11 ml of the lysing solution was aerated and the two layers were gently mixed. The resulting solution was centrifuged at 100,000 g for 1 hour and 5M NaCl was added to the supernatant to give a final concentration of 0.1M (870 μΐ 5M NaCl in 43.5 ml supernatant) and equilibrated 2 x 12 with A buffer. cm on a DE32 column. The β-subunit of the reverse transcriptase does not bind to the column, but a substantial part of the DNA primerase and nucleic acids that interfere with the following chromatography do.

The column was washed with Buffer A until the unbound material was completely removed (eluent extinction A28 0 <0.1) and the effluent solution (140 mL) was dialyzed 3 x 4 L (3 x 1 L of Buffer B). was applied to a 2 x 20 cm phosphocellulose (P11) column, which was washed with B buffer (200 mL in our example) until extinction of A 1 < 0.1, and eluted with a 0-1.0 M gradient (B). buffer), 4 ml fractions were collected.

Fractions containing 3 μΐ were tested and fractions containing reverse transcriptase activity between 0.25-0.33 M were collected and dialyzed against 3x21 C buffer for 3x8 hours.

After dialysis, the material was loaded onto a 0.6 x 6 cm Heparin-Sepharose column, the unbound material was washed with buffer C and the enzyme was eluted from the column with 0.01.0 M KCl. 0.6 ml fractions were collected and assayed for reverse transcriptase activity in 2 μΐ.

The active fractions (about 2.0 mL) were dialyzed against D-buffer for 18 hours

The total amount of enzyme after purification and concentration is about 10,000 U (800 ml of the original bacterial culture). The peak fraction contains about one third of the total enzyme activity. After concentration, its activity is 20-25 U / μΙ.

The resulting enzyme has the following characteristics:

The preparation was able to synthesize cDNA in the standard reaction mixture for a minimum of 2 hours, in contrast to the control Boehringer enzyme (RT Lot No: 10396024-25 / Oct. 86) which synthesized more cDNAs over a 20 minute incubation period. during the lesson. This data, as well as the unchanged gel image of RNA incubated with our preparation for 4 hours (Fig. 4), confirm that the purified recombinant reverse transcriptase is free of a commercially available reverse transcriptase preparation isolated from a number of viruses. from non-specific nucleases.

Recombinant reverse transcriptase is capable of poly dT synthesis in the presence of both Mg ** and Mn ** ions in the rA / dT ₁₃ primer system.

The optimal concentrations of these ions are 3 and 2 mM, respectively. Slightly lower reaction rates can be measured in the presence of 6 mM Mg ** or Mn **.

The enzyme, unlike DNA polymerase, also functions in the polyCn / oligo dG primer-template system, but only in the presence of Mn **. The activity of this substrate is about 25% of that of the classic poly rA / dT substrate. The measurement data obtained in the above system are in complete agreement with other vital literature

202,280 with reverse transcriptase data (Gerard et al., Biochemistry, 13,1632 (1974)).

In the PoliA ⁺ mRNA-oligo dT primer-template system (i.e., cDNA synthesis), the Mg ⁺⁺ optimum of the enzyme is 6mM, the Mn ** optimum is 2mM. Under these conditions, 7.5 U enzymes incorporate 200 and 400 pmol / Ή / 1,5 of 0.12 pg mRNA in 1.5 hours, which corresponds to the generation of 0.8-1.6 nmol cDNA nucleotides. The resulting product had a size of 200-1600 nucleotides for polyA * Drosophila mRNA, and 400-7000 nucleotides using the significantly longer polyA * rat liver hnRNA (Figure 5).

The data in this example justifies:

1. The protein produced by the clone has the same enzyme activity as the viral reverse transcriptase.

2. The preparation shall be free from any interfering impurities which would interfere with its molecular biological use (nucleases).

3. The recombinant reverse transcriptase is capable of template-transcribing RNA with the appropriate primer, however long, but at least 7,000 nucleotides in length, which is much longer than the average mRNA.

Claims (4)

1. A method of producing a reverse transcriptase of Rous sarcoma virus, comprising the step of ligating a Rous sarcoma virus-derived pol gene encoding a reverse transcriptase from pSRA2 into a plasmid pUC9, transforming the ligand with E. coli by selection. sequence repairs, expression, binding of the mRNA to the ribosome, and sequences providing both the translational initiator and terminator sequences are transformed into recombinant, E. coli, bacterial cells are lysed, and the extracted crude reverse transcriptase is optionally purified. Priority: 04.09.1989. 2. The method of claim 1, wherein the E. coli strain is E. coli HB101. Priority: 04.09.1989. The method according to any one of claims 1 or 2, characterized in that the viral pol gene region between the PstI and XhoI sites of plasmid pSRA2 is cloned into the plasmid pUC9, which has been opened by Psfl and SalI enzymes. Priority: 04.09.1989. 4. A process according to claim 1, wherein the purification is carried out by ion exchange chromatography. Priority: 04.09.1989.