RU2845454C1 - Method of producing tms-rev reverse transcriptase - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to recombinant production of reverse transcriptase, and can be used to produce modified reverse transcriptase from Moloney murine leukaemia virus (MMLV). Reverse transcriptase is obtained by a recombinant method comprising obtaining a nucleotide sequence encoding said reverse transcriptase, obtaining an expression plasmid containing said nucleotide sequence, expression of said reverse transcriptase in E.coli cells, isolating and purifying said reverse transcriptase, as a result of which said reverse transcriptase is obtained in a buffer solution containing 20 mM MES, 100 mM NaCl, 1 mM EDTA, 20 % glycerine, pH 6.5, 25 mM MgCl₂.

EFFECT: invention enables to obtain reverse transcriptase, having high revertase activity, with stable high output of the enzyme.

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Description

The invention relates to the field of genetic engineering and biotechnology, in particular to a method for producing a preparation of the reverse transcriptase enzyme in the form of a recombinant protein in E. coli cells with a high yield of the enzyme in soluble form.

Methods of enzymatic analysis of nucleic acids are an integral part of modern fundamental research and applied disciplines, including medical and veterinary diagnostics. Among them, an important place is occupied by enzymatic RNA synthesis using a DNA matrix, i.e. reverse transcription, which allows analyzing a total set of RNA or individual RNA molecules in different cellular samples. From the point of view of fundamental research, reverse transcription makes it possible to convert RNA into a more convenient for analysis and more stable form of nucleic acid - complementary DNA, cDNA. The key component of reverse transcription are enzymes, namely RNA-dependent DNA polymerases or reverse transcriptases, capable of synthesizing DNA using an RNA matrix. In the context of practical use of reverse transcriptases, a number of their enzymatic characteristics are of great importance, such as thermal stability, specific optimum operating temperature, accuracy, processivity and resistance to inhibitors. Improving these properties increases the quantity and quality of synthesized complementary DNA, which in turn facilitates subsequent cDNA analysis.

Reverse transcriptase (RT) was first discovered in 1970 and isolated from retroviruses. The name "retrovirus" comes from the ability of these viruses to replicate in host cells by converting their RNA genomes into DNA. It was established that the process of reverse transcription is only possible due to the enzyme reverse transcriptase [Mizutani S, Boettiger D, Temin HM (1970) A DNA-dependent DNA polymerase and a DNA endonuclease in virions of rous sarcoma virus. Nature 228:424-427].

Reverse transcriptase (RT) has the activity of RNA- and DNA-dependent DNA polymerase and RNase H. The RT enzyme is a monomeric or dimeric protein with two active centers in the DNA polymerase and RNase H endonuclease domains.

RTs from Moloney Murine Leukemia Virus (MMLV) and Avian Myeloblastosis Virus (AMV) have been most widely used for cDNA synthesis and RNA amplification reactions due to their high catalytic activity and fidelity. MMLV RT is a 75 kDa monomer, while AMV RT is a heterodimer consisting of a 63 kDa a subunit and a 95 kDa b subunit. The structure of MMLV RT includes finger, palm, thumb, junction, and RNase H domains. The a subunit of AMV RT includes these five domains, and the b subunit includes five domains and a C-terminal integrase domain. Reverse transcription and RNase H cleavage occur simultaneously when RT is bound to a single template primer.

The thermal stability of reverse transcriptase was markedly improved by eliminating the RNase H domain activity. As a result, the reaction temperature of MMLV RT and AMV RT for reverse transcription increased from 37-45°C to 50-60°C, suggesting that the two activities are not completely functionally independent [Yasukawa, K., Nemoto, D., and Inouye, K. (2008) Comparison of the thermal stabilities of reverse transcriptases from avian myeloblastosis virus and Moloney murine leukaemia virus. J. Biochem. 143, 261-268]. In cDNA synthesis and RNA amplification, elevated reaction temperatures are desirable because they destabilize the secondary structure of RNA and reduce nonspecific primer binding.

The most preferred and widely used enzyme in molecular research or laboratory work due to its high catalytic activity and accuracy is RT from MMLV [Nishimura K, Yokokawa K, Hisayoshi T, Fukatsu K, Kuze I, Konishi A, Mikami B, Kojima K, Yasukawa K (2015) Preparation and characterization of the RNase H domain of moloney murine leukemia virus reverse transcriptase. Protein Expr Purif 113:44-50. doi: https:// doi. org/ 10. 1016/j. pep.2015. 04. 012]. Some studies have shown that the thermal stability and efficiency of MMLV-RT can be improved by using site-directed mutagenesis, rational design and production of recombinant enzymes using E. coli expression system [Konishi A, Ma X, Yasukawa K (2014) Stabilization of moloney murine leukemia virus reverse transcriptase by site-directed mutagenesis of the surface residue Val433. Biosci Biotechnol Biochem 78:147–150; Mizuno M, Yasukawa K, Inouye K (2010) Insight into the mechanism of the stabilization of moloney murine leukemia virus reverse transcriptase by eliminating RNase H activity. Biosci Biotechnol Biochem 74:440–442]. The potent RNase H activity is useful in PCR to degrade RNA molecules in the RNA-DNA duplex during the first PCR cycles. However, when using long RNA templates, RNase H activity may degrade the RNA early, resulting in shortened cDNA.

RT enzyme plays an important role as a molecular tool in RT-PCR, RNA sequencing, gene expression analysis, cDNA cloning and any other molecular approach that involves cDNA synthesis from RNA molecules [Costa C, Gimenez-Capitan A, Karachaliou N, Rosell R (2013) Comprehensive molecular screening: from the RT-PCRto the RNA-seq. Transl Lung Cancer Res 2:87–91. doi: https:// doi. org/10. 3978/j. issn. 2218-6751. 2013. 02. 05].

Reverse transcription-PCR (RT-PCR) is a common laboratory technique that uses reverse transcriptase to create cDNA from target RNA sequences. RT-PCR is performed as a two- or one-step assay. In two-step assays, reverse transcription is performed first, followed by PCR. In a one-step assay, reverse transcription and PCR are performed in the same tube in the same buffer, using a single- or dual-enzyme system. One-step assays are faster and minimize the risk of sample contamination. However, one-step assays are more difficult to develop because buffer conditions must be adjusted to maximize the speed and efficiency of both the cDNA and PCR steps using the same set of conditions. Studies of RT activity have led to the development of oligonucleotide binding models. [V.M. Wohrl, R. Krebs, R.S. Goody, T. Restle, Refined model for primer/template binding by HIV-1 reverse transcriptase: pre-steady-state kinetic analyzes of primer/template binding and nucleotide incorporation events distinguish between different binding modes depending on the nature of the nucleic a, J. Mol. Biol. 292 (1999) 333 344, https://doi.org/10.1006/JMBI. 1999.3057].

Production of proteins as well as enzymes in sufficient quantities is key for their study or use in various molecular applications. Escherichia coli is a preferred host for the production of recombinant proteins given its rapid growth, ease of manipulation, and cost-effectiveness. Currently, many proteins and enzymes of commercial interest are produced in E. coli [Baeshen MN, Al-Hejin AM, Bora RS, Ahmed MM, Ramadan HA, Saini KS, Baeshen NA, Redwan EM (2015) Production of biopharmaceuticals in E. coli : current scenario and future perspectives. J Microbiol Biotechnol 25:953–962]. However, using E. coli as a tool for the production of recombinant enzymes poses two major challenges: 1) poor expression of the foreign gene and 2) the ability of recombinant proteins to be overexpressed. The use of different plasmid vectors, selection of an optimal host strain, or co-expression with other genes can increase the yield of the target protein and promote correct folding of the molecule [Redwan ELRM (2006) Arab J Biotechnol 9(3):493–510].

In this regard, the task of developing the simplest, fastest and most cost-effective method for obtaining reverse transcriptase without the use of complex methods and expensive components, as well as without the use of additional labels in the enzyme sequence, with a high yield of the target product and activity that meets the requirements for use in various molecular methods, is relevant.

The technical task of the proposed invention is to develop an effective and rapid method for obtaining reverse transcriptase in soluble form, with a high yield of the target protein when expressed in E. coli while maintaining or increasing its specific activity. At the same time, the preparation should be suitable for use in the RT-PCR system and other applications.

The technical result is achieved by modifying the nucleotide and amino acid sequence of the wild-type MMLV reverse transcriptase, selecting a vector for expression and varying the cultivation conditions, as well as a rapid and effective method for purifying the reverse transcriptase enzyme.

Thus, the claimed method for obtaining a recombinant reverse transcriptase preparation is based on the transformation of competent E. coli cells with an expression plasmid encoding the target protein, with subsequent cultivation of the transformed producer cells under conditions that ensure the expression of the said recombinant reverse transcriptase in a Luria-Bertani (LB) nutrient medium with the addition of ampicillin and the use of isopropyl-pD-thiogalactoside (IPTG) as an inducer for the expression of the target protein, the isolation of the target protein from the soluble fraction and the performance of a two-stage chromatographic purification.

The TMS nucleotide sequence SEQ ID NO:1 encoding the TMS-Rev amino acid sequence SEQ ID NO:2 was obtained by introducing the Q263R and R449C mutations into the wild-type gene (GenBank: AF033811.1, region 1978-3990 bp) encoding the wild-type MMLV reverse transcriptase from Moloney murine leukemia virus (EC 2.7.7.49, GenBank: AAC82568.2, region 661-1330 aa). For this purpose, primers for mutagenesis carrying target substitutions are used: Q263R F SEQ ID NO: 3, Q263R R SEQ ID NO: 4 and R449C F SEQ ID NO: 5, R449C R SEQ ID NO: 6. Substitutions in the nucleotide sequence of the wild-type gene (GenBank: AF033811.1, region 1978-3990 bp) are introduced by mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (Agilent, USA) or any other commercial kit in accordance with the manufacturer's instructions. For subsequent cloning into the expression vector, the NdeI restriction endonuclease site is introduced at the 5' end of the nucleotide sequence obtained as a result of mutagenesis, and XhoI at the 3' end using amplification using the TMS-for-Nde SEQ ID NO 7 and TMS-rev-Xho SEQ ID NO 8 primers.

As a result, the nucleotide sequence TMS SEQ ID NO:l is obtained, which is used to obtain expression constructs.

To clone the fragment carrying the target gene, restriction of the amplicon containing the target nucleotide sequence TMS SEQ ID NO: 1 and the vector for expression of the pET system (Novagen, USA) is carried out. Restriction of plasmid DNA is carried out for 1 hour in the appropriate buffer solution at 37° in 20 μl of the reaction mixture. The reaction is carried out according to the standard protocol recommended by the manufacturer for restriction enzymes NdeI and XhoI (NEB, USA). For the hydrolysis reaction of 1-2 μl of DNA with a concentration of 300 ng, 2-4 units of each restriction enzyme are taken. The efficiency of the reaction is assessed by electrophoresis in 1% agarose gel. Then, ligation of the target insert and vector DNA is carried out and transformation of the ligase mixture into E. coli DH10B cells (Thermo Fisher Scientific, USA) is carried out. Clones containing the target insert can be identified by PCR with universal primers T7-forward SEQ ID NO:9 and T7-reverse SEQ ID N0:10 (Genterra, Russia). The correctness of insertion and the presence of the target sequence are determined using Sanger sequencing. As a result, the expression plasmid pETTMS is obtained, containing the nucleotide sequence SEQ ID NO: 1.

Expression is carried out in E. coli cells on Luria-Bertani (LB) nutrient medium. The pETTMS expression construct containing the TMS gene sequence SEQ ID NO:1 encoding the TMS-Rev reverse transcriptase, which corresponds to the amino acid sequence SEQ ID NO:2, is introduced into E. coli Rosetta De3 cells (Thermo Fisher Scientific, USA) by electroporation. As a result, E. coli Rosetta De3/pET_TMS producer cells are obtained.

Experimental expression is carried out on Luria-Bertani (LB) nutrient medium containing 10 g/l tryptone, 5 g/l yeast extract and 10 g/l sodium chloride in the presence of 100 μg/ml ampicillin; isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mM is used as an expression inducer. Protein synthesis in cells was carried out for 23 hours after addition of the inducer. The target protein is isolated from the soluble protein fraction, purification is carried out by chromatography in two stages.

To obtain a starting culture, E. coli Rosetta De3/pET TMS producer cells are grown at 37°C for 12-16 hours, then the cells are transferred to a fresh LB nutrient medium containing 100 μg/ml ampicillin and 0.2% glucose. Also, after dilution, the producer cells are cultured at 37°C until an optical density of OD600-0.7-1.0 o.u is achieved, after which IPTG is added to the nutrient medium and the cell culture is incubated for 23 hours at a temperature of 23°C.

The cells are resuspended in a buffer solution containing 20 mM Mes, 0.1% PMSF, pH 6.5 with the addition of 25 mM MgCl ₂ and treated with ultrasound to disintegrate the cells. Then centrifugation is carried out at 6000 g for 30 minutes. As a result, the presence of the TMS-Rev protein with the amino acid sequence SEQ ID NO: 2, corresponding to the calculated mass of 73 kDa (Fig. 1 and Fig. 2) was shown in the resulting solution using electrophoresis under denaturing conditions.

Chromatographic purification is carried out in two stages: metal-chelate chromatography is performed at the first stage, and cation-exchange chromatography at the second stage. The target protein is eluted with a linear gradient in a buffer solution containing 20 mM MES, 500 mM imidazole, pH 6.5 in the case of metal-affinity chromatography, and 20 mM MES, 1 M NaCl, pH 6.5, with the addition of 25 mM MgCl ₂ , - for cation-exchange chromatography (Fig. 3, Fig. 4). The final stage of purification is gel filtration chromatography on G-25 sorbent (GE Healthcare). As a result of the developed purification method, we obtain an enzyme with a purity of more than 95% (Fig. 5).

As a result of expression and chromatographic purification, we obtain a TMS-Rev reverse transcriptase preparation of the following composition: recombinant TMS-Rev reverse transcriptase enzyme, containing a recombinant protein with the amino acid sequence SEQ ID NO: 2, encoded by the nucleotide sequence TMS SEQ ID NO: 1, in a buffer solution containing 20 mM MES, 100 mM NaCl, 1 mM EDTA, 20% glycerol, pH 6.5, with the addition of 25 mM MgCl ₂ .

The claimed invention is the result of work within the framework of the development of enzymes used in various methods of molecular diagnostics, carried out at the Federal Budgetary Scientific Institution Central Research Institute of Epidemiology of Rospotrebnadzor (Moscow, Russia).

The claimed solution is illustrated in Fig. 1-6, where

Fig. 1. Electropherogram of fractions containing the recombinant protein reverse transcriptase TMS-Rev, obtained by expression in E. coli Rosetta De3 cells, where: M-molecular mass marker 26619 (Thermo Fisher Scientific, USA);

1 total cell lysate of the producer strain;

2 clarified cell lysate of the producer strain;

3 - fraction of insoluble proteins of the producer strain.

Fig. 2. Electropherogram of fractions containing the recombinant protein reverse transcriptase TMS-Rev, obtained by expression in E. coli Rosetta De3 cells, where:

M - molecular mass marker 26619 (Thermo Fisher Scientific, USA);

4 total cell lysate of the producer strain Rosetta De3 before induction of protein biosynthesis;

5 - total cell lysate of the producer strain Rosetta De3 after induction of protein biosynthesis;

6 - clarified cell lysate of the producer strain Rosetta De3 after induction of protein biosynthesis;

7 - cell pellet of the Rosetta De3 producer strain after induction of protein biosynthesis.

Fig. 3. Chromatographic profile of purification of recombinant reverse transcriptase protein on Masgorger High S support at pH 6.5; where the numbers 8-11 indicate the stages of the process, where:

8 application

9 washing away unbound proteins

10 - 11 - elution with a linear gradient of a solution containing 1 M sodium chloride, with 11 being the fraction of the target enzyme.

Fig. 4. Electropherogram of chromatographic fractions containing recombinant reverse transcriptase protein obtained during purification on Masgorger High S support, where:

M - molecular mass marker 26619 (Thermo Fisher Scientific, USA);

12 application;

13 - slip;

14-17 - fractions of ballast cell proteins at 650 mM sodium chloride; 18-21 - fractions of eluate at 700 mM sodium chloride;

22 - eluate fractions at 1 M sodium chloride.

Fig. 5. Electropherogram of the chromatographic fraction containing the recombinant reverse transcriptase protein obtained during purification on G-25, where: M is the molecular weight marker 26619 (Thermo Fisher Scientific, USA);

23 - total fraction of the target enzyme.

Fig. 6. Results of comparison of the work of TMS-Rev from the Rosetta De3/TMS strain and RT-Promega by the RT-PCR method on a CFX96 amplifier, where: A - FAM channel - RdRp gene of SARS-CoV-2; B - JOE/HEX channel - E gene of SARS-CoV-2; C - ROX - N gene of SARS-CoV-2;

D - Cy5 - VKO;

pink curves are the Promega enzyme;

Red curves of TMS-Rev from Rosetta De3 strain.

The implementation of the claimed invention is illustrated by the following examples:

Example 1. Obtaining the nucleotide sequence of reverse transcriptase by PCR and its cloning.

The previously obtained wild-type reverse transcriptase enzyme gene EC 2.7.7.49 (GenBank: AF033811.1, region 1978-3990 bp), encoding wild-type MMLV reverse transcriptase from Moloney murine leukemia virus (EC 2.7.7.49, GenBank: AAC82568.2, region 661-1330 aa), was used as a template. The following mutations were introduced to increase the temperature stability, enzyme solubility, and RNA stability in the RNA/DNA complex: Q263R and R449C. For this purpose, primers for mutagenesis carrying target substitutions were used: Q263R F SEQ ID NO: 3, Q263R R SEQ ID NO: 4 and R449C F SEQ ID NO: 5, R449C R SEQ ID NO: 6. Substitutions in the nucleotide sequence of the wild-type gene (GenBank: AF033811.1, region 1978-3990 bp) were introduced by mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (Agilent, USA). For subsequent cloning into expression vectors, a restriction endonuclease site NdeI was introduced at the 5' end of the nucleotide sequence SEQ ID NO:1, and XhoI at the 3' end using amplification using primers TMS-for-Nde SEQ ID NO 7 and TMS-rev-Xho SEQ ID NO 8. The presence of target mutations was confirmed using Sanger sequencing.

As a result, the nucleotide sequence TMS SEQ ID NO:l was obtained, which was subsequently used to obtain expression constructs.

Example 2. Obtaining expression constructs encoding reverse transcriptase.

To clone the fragment carrying the target gene, restriction of the amplicon containing the target nucleotide sequence TMS SEQ ID NO: 1 and the vector for expression of the pET system (Novagen, USA) was performed. Restriction of plasmid DNA was performed for 1 hour in the appropriate buffer solution at 37° in 20 μl of the reaction mixture. The reaction was carried out according to the standard protocol recommended by the manufacturer for restriction enzymes NdeI and XhoI (NEB, USA). For the hydrolysis reaction of 2 μl of DNA with a concentration of 300 ng, 4 units of restriction enzyme were taken. The efficiency of the reaction was assessed by electrophoresis in 1% agarose gel. Then, ligation of the target inserts and vector DNA was performed and the ligase mixture was transformed into E. coli DH10B cells.

(Thermo Fisher Scientific, USA). Clones containing the target insert were identified by PCR with universal primers T7-forward SEQ ID NO:9 and T7-reverse SEQ ID N0:10 (Genterra, Russia). The correctness of insertion and the presence of the target sequence were determined using Sanger sequencing. As a result, the expression plasmid pET TMS containing the nucleotide sequence SEQ ID NO:1 was obtained.

Example 3. Expression of TMS-rev reverse transcriptase in E.coli cells.

The expression construct pET TMS, containing the TMS gene sequence SEQ ID NO:1, encoding the reverse transcriptase TMS-Rev, corresponding to the amino acid sequence SEQ ID NO:2, was introduced into E. coli Rosetta De3 cells (Thermo Fisher Scientific, USA) by electroporation. As a result, E. coli Rosetta De3/pET_TMS producer cells were obtained.

Experimental expression was performed on Luria-Bertani (LB) nutrient medium containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L sodium chloride in the presence of 100 μg/mL ampicillin; isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5 mM was used as an expression inducer. Protein synthesis in cells was carried out for 23 hours after addition of the inducer. The target protein was isolated from the soluble protein fraction, and purification was performed by chromatography in two stages.

To obtain the starting culture, E. coli Rosetta De3/TMS-rev producer cells were grown at 37°C for 12 hours, then the cells were transferred to fresh LB nutrient medium containing 100 μg/ml ampicillin and 0.2% glucose. Also, after dilution, the producer cells were cultured at 37°C until an optical density of OD600 of 0.7-1.0 o.u was achieved, after which IPTG was added to the nutrient medium and the culture was incubated for 23 hours at 23°C.

The cells were resuspended in a buffer solution containing 20 mM Mes, 0.1% PMSF, pH 6.5 with the addition of 25 mM MgCl ₂ and treated with ultrasound to disintegrate the cells. Then centrifugation was performed at 6000 g for 30 minutes. As a result, the presence of the TMS-Rev protein with the amino acid sequence SEQ ID NO: 2, corresponding to the calculated mass of 73 kDa was shown in the resulting solution using electrophoresis under denaturing conditions (Fig. 1 and Fig. 2).

Chromatographic purification was performed in two stages: metal-chelate chromatography was performed in the first stage, and cation-exchange chromatography was performed in the second stage. The target protein was eluted with a linear gradient in a buffer solution containing 20 mM MES, 500

mM imidazole, pH 6.5 for metal affinity chromatography, and 20 mM MES, 1 M NaCl, pH 6.5, with the addition of 25 mM MgCl ₂ , for cation exchange chromatography (Fig. 3, Fig. 4). The final purification step is gel filtration chromatography on G-25 sorbent (GE Healthcare). As a result, the enzyme with a purity of more than 95% was obtained (Fig. 5).

As a result of expression and chromatographic purification, a preparation of TMS-Rev reverse transcriptase of the following composition was obtained: recombinant TMS-Rev reverse transcriptase enzyme containing the amino acid sequence SEQ ID NO: 2, encoded by the nucleotide sequence TMS SEQ ID NO: 1, in a buffer solution containing 20 mM MES, 100 mM NaCl, 1 mM EDTA, 20% glycerol, pH 6.5, with the addition of 25 mM MgCl ₂ .

Example 4. Testing the activity of the obtained enzyme preparation under RT-PCR conditions.

The catalytic characteristics of the obtained enzyme were tested under RT-PCR reaction conditions. The properties of the obtained preparation were compared with a commercially available reference enzyme - M-MLV Reverse Transcriptase (Promega, USA) at a working concentration recommended by the manufacturer - 200 U/ml.

The testing was performed using a laboratory technique for detecting various fragments of the SARS-CoV-2 genome using three different primer-probe systems for different target genes: RdRp gene of RNA-dependent RNA polymerase, E gene of the viral envelope protein (Envelope) and N gene of the nucleoprotein. In all experiments, the commercial enzyme M-MLV Reverse Transcriptase (Promega, USA) was used as a reference enzyme. Detection was performed in four channels: FAM - RdRp gene, JOE/HEX - E gene, ROX N gene, Cy5 detection of the internal control sample (ICS). RNA-protected positive control samples (PCS) with a concentration of ¹⁰³ cop/ml were used as detection samples. The reaction was carried out in a reaction mixture of the following composition: 50 mM Tris-HCl (pH 8.3), 17 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgCl, 1.4 mM of each dNTP, 0.2 μM of primers and probe, 2.5 tM 1-thioglycerol. The reaction mixture also contained 1 U of active TaqF polymerase (AmpliSens, Russia). The amplification program included a reverse transcription step for 20 min at 50°C, a denaturation step for 15 min at 95°C, then 40 amplification cycles: 95°C - 10 sec, 60°C - 10 sec, 72°C - 10 sec. Detection of fluorescent signals was performed at the end of each amplification cycle.

The results of the experiments show that the TMS-Rev reverse transcriptase enzyme preparation containing the recombinant protein with the amino acid sequence SEQ ID NO: 2, encoded by the nucleotide sequence SEQ ID NO: 1, in a buffer solution containing 20 mM MES, 100 mM NaCl, 1 mM EDTA, 20% glycerol, pH 6.5, with the addition of 25 mM MgCl ₂ has catalytic activity, can be used in RT-PCR reactions, and is also comparable in its level of efficiency with the comparison enzyme M-MLV Reverse Transcriptase (Promega, USA) (Fig. 6).

Thus, the claimed invention makes it possible to obtain a reverse transcriptase enzyme in E. coli cells with a consistently high yield of the enzyme, characterized by a sufficiently high reverse transcriptase activity, which makes it possible to use it in RT-PCR reactions associated with the detection of RNA of organisms pathogenic to humans.

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</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPL

IIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREV

NKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRL

PQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKK

AQICRKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLT

KTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKK

LDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDCWLSNARMTHYQALLLDTD

RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGA

AVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEG

KEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL</INSDS

eq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="3">

<INSDSeq>

<INSDSeq_length>28</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..28</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier>

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>TMS-Rev</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tacctctgaaagcaacgtctacccccgt</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="4">

<INSDSeq>

<INSDSeq_length>28</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..28</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier>

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>TMS-Rev</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>acgggggtagacgttgctttcagaggta</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="5">

<INSDSeq>

<INSDSeq_length>26</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..26</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier>

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>TMS-Rev</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tcactatcaggccttgcttttggaca</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="6">

<INSDSeq>

<INSDSeq_length>26</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..26</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier>

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>TMS-Rev</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tgtccaaaagcaaggcctgatagtga</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="7">

<INSDSeq>

<INSDSeq_length>27</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..27</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier>

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>TMS-Rev</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gtcgtcatatgaccctaaatatagaag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="8">

<INSDSeq>

<INSDSeq_length>25</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..25</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier>

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>TMS-Rev</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctcgagttagaggagggtagaggtg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="9">

<INSDSeq>

<INSDSeq_length>23</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..23</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier>

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>TMS-Rev</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>aattaatacgactcactataggg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="10">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier>

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>TMS-Rev</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tatgctagttattgctcagcgg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (8)

1. A method for producing TMS-Rev reverse transcriptase as a recombinant protein in E.coli cells with a high yield of the enzyme in soluble form, comprising: (i) obtaining the nucleotide sequence of SEQ ID NO: 1; (ii) obtaining the expression plasmid pET_TMS containing the nucleotide sequence of SEQ ID NO: 1, wherein the amplicon containing the target nucleotide sequence of SEQ ID NO: 1 and the vector for expression of the pET system are sequentially digested, the target insert and vector DNA are ligated, and the ligase mixture is transformed into E. coli DH10B cells; (iii) expression of TMS-Rev reverse transcriptase from Molonia murine leukemia virus (MMLV) in E. coli cells on Luria-Bertani nutrient medium in the presence of ampicillin, wherein the expression construct containing the nucleotide sequence of SEQ ID NO: 1 is introduced into E. coli Rosetta De3 cells by electroporation, isopropyl-β-D-thiogalactoside is used as an expression inducer, protein synthesis in the cells is carried out for 23 hours at a temperature of 23°C after addition of the inducer, the target protein is isolated from the soluble protein fraction, and purification is carried out by chromatography in two stages; (iv) obtaining a TMS-Rev reverse transcriptase preparation of the following composition: a recombinant protein with the amino acid sequence of SEQ ID NO: 2, encoded by the nucleotide sequence of SEQ ID NO: 1, in a buffer solution containing 20 mM MES, 100 mM NaCl, 1 mM EDTA, 20% glycerol, pH 6.5, 25 mM MgCl ₂ . 2. A method for producing TMS-Rev reverse transcriptase according to claim 1, wherein the nucleotide sequence of SEQ ID NO: 1 is obtained by introducing the Q263R and R449C mutations into the wild-type gene encoding MMLV reverse transcriptase, using mutagenesis primers carrying target substitutions SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and at the ends of the nucleotide sequence of SEQ ID NO: 1 using primers SEQ ID NO 7 and SEQ ID NO 8, restriction endonuclease sites NdeI are introduced at the 5`-end, XhoI at the 3`-end using primers TMS-for-Nde SEQ ID NO 7 and TMS-rev-Xho SEQ ID NO 8. 3. A method for obtaining TMS-Rev reverse transcriptase according to claim 1, wherein the restriction of plasmid DNA is carried out for 1 hour in a buffer solution at 37°C in 20 μl of the reaction mixture, followed by ligation of the insert containing the reverse transcriptase gene into the pET series expression vector, to obtain the pET_TMS expression plasmid construct. 4. The method for obtaining TMS-Rev reverse transcriptase according to claim 1, wherein the expression in step (iii) is carried out on a Luria-Bertani nutrient medium containing 10 g/l tryptone, 5 g/l yeast extract and 10 g/l sodium chloride, in the presence of 100 μg/ml ampicillin, and isopropyl-β-D-thiogalactoside with a final concentration of 0.5 mM is used as the expression inducer.