RU2846583C1 - Recombinant plasmid dna pb2k_t5_sumo_hrv_3c, providing synthesis of chimeric protein sumo-hrv_3c in escherichia coli cells and recombinant escherichia coli krx/pb2k_t5_sumo_hrv_3c strain, which produces chimeric protein sumo-hrv_3c used to produce recombinant picornaine 3c a28 enzyme used to cleave fused proteins and affinity tags - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: what is presented is recombinant plasmid DNA pB2k_T5_SUMO_HRV_3C providing synthesis of chimeric protein SUMO-HRV_3C in E. coli cells having nucleotide sequence SEQ ID NO: 1 with size of 4193 bp. Also disclosed is recombinant E. coli strain KRX/pB2k_T5_SUMO_HRV_3C, obtained by transformation of E. coli cells of recipient strain KRX said recombinant plasmid DNA pB2k_T5_SUMO_HRV_3C and providing synthesis of chimeric protein SUMO-HRV_3C with calculated molecular weight 32.5 kDa.

EFFECT: invention enables to obtain recombinant protein 3C (picornaine 3C of rhinovirus A28) for splitting fused proteins and splitting affinity marks.

2 cl, 9 dwg, 1 tbl, 5 ex

Description

The group of inventions relates to the recombinant plasmid DNA pB2k_T5_SUMO_HRV_3C, providing the synthesis of the chimeric protein SUMO-HRV_3C in Escherichia coli ( E. coli) cells, the recombinant E. coli strain KRX/pB2k_T5_SUMO_HRV_3C, producing the chimeric protein SUMO-HRV_3C and to the recombinant enzyme picornaine 3C of the rhinovirus A28 without affinity labels, which can be used in the pharmaceutical industry, biotechnology and fundamental research, in particular, in the production of recombinant proteins for X-ray structural analysis.

Human rhinoviruses (HRV) are nonenveloped viruses of the Picornaviridae family of the Enterovirus genus, whose genome is represented by single-stranded RNA of positive polarity, and are among the most common viral pathogens causing respiratory diseases worldwide. One of the main rhinovirus proteases is picornain 3C, which is a monomeric protein with a molecular weight of about 20 kDa. This enzyme is necessary for post-translational proteolysis of viral precursor polyproteins and for proteolytic degradation of cellular proteins involved in cellular transcriptional processes and antiviral defense [1].

The absence of cellular homologues of protease 3C allows this enzyme to be used as a potential target for the development of antiviral compounds. However, the task is complicated by the significant diversity of rhinoviruses: more than 100 serotypes are currently known. In order to study parallel evolutionary changes in the structure of viral proteolytic enzymes, it is necessary to conduct comprehensive studies of the properties of picornaine 3C of various serotypes.

Today, it is known that most buffers and auxiliary compounds used in the production of recombinant proteins do not have a significant effect on the activity of picornaine 3C, which is important for the biotechnology industry [2].

The 3C enzyme is a cysteine protease and has a high level of enzymatic activity, including at a temperature of 4°C, and strict specificity of the amino acid sequence of the recognition site Leu-Gln-Ala-Ile-Phe-Gln↓Gly-Pro (hydrolyzes the peptide bond between glutamine and glycine), making it convenient to use for removing affinity tags and proteolytic cleavage of fused recombinant proteins.

A decision is known on a Chinese patent (CN108795964, IPC C12N 15/70, published on 13.11.2018) [4], where a variant of a plasmid construct was developed based on the PGEX-4T-1 vector, providing expression in E. coli cells of a fused recombinant picornaine 3C of the rhinovirus B14, the gene of which is located in the same open reading frame with the glutathione S-transferase (GST) gene of S. japonicum . Purification of the recombinant protein was carried out using affinity and size-exclusion chromatography methods. There is also a commercially available recombinant protein PreScission, consisting of a recombinant protease 3C of the rhinovirus B14, fused with a 6xHis tag.

A decision on a Chinese patent (CN116949019, IPC C12N 15/70, published on 27.10.2023) [5] is known, which describes a protocol for obtaining a recombinant enzyme 3C of rhinovirus B14 fused with 6xHis and the Leadesseq affinity tag (a fragment of a sequence from a spider silk protein). The developed method with the Leadesseq tag allows achieving a yield of recombinant picornaine 3C 2.3 times higher than using the GST affinity tag, with high purity and preserved enzymatic activity.

The closest analogue (prototype) is the solution proposed by a group of researchers from the National Institute of Biotechnology and Genetic Engineering of Pakistan (Blom N. et al. Cleavage site analysis in picornaviral polyproteins: discovering cellular targets by neural networks //Protein Science. - 1996. - Vol. 5. - No. 11. - Pp. 2203-2216.) [2]. The picornaine 3C gene of rhinovirus B14 was cloned in the pKLD116 vector, containing sequences of the 8xHis tag, linker, maltose-binding protein (MBP) and the cleavage site of TEV protease (Tobacco Etch Virus). The work compared the enzymatic activity of recombinant proteases 3C and TEV at temperatures of 25°C and 4°C, and assessed the effect of various detergents and buffer components used in the purification of recombinant proteins. It was shown that the efficiency of the enzyme at a temperature of 4°C is higher for protease 3C than for the TEV protein, and protease 3C is insensitive to most of the tested buffer solutions, which is an advantage over other proteases in the use of the enzyme 3C for the cleavage of fusion proteins and affinity tags, including in large-scale production of recombinant proteins.

The listed analogues and prototype use the well-studied and used in purification of recombinant proteins picornain 3C serotype B14. The claimed application for invention presents protease 3C of rhinovirus serotype A28, which, according to data obtained using the Basic Local Alignment Search Tool (BLAST) [5], is no more than 52% identical to picornain 3C serotype B14. To date, the NCBI databases do not have an annotated variant of protease 3C serotype A28, the properties of recombinant analogues have not been studied or compared with already known prototypes.

A disadvantage of the analogue according to the Chinese patent (CN108795964, IPC C12N15/70, published on 13.11.2018, [4]) is the presence of GST and MBP affinity tags, which, compared to the SUMO affinity tag, increase the solubility of the target protein with less efficiency [6]. The Leadesseq affinity tag (CN 116949019, published on 27.10.2023) [5] has been used recently, so its properties have not been sufficiently studied.

A common drawback of analogues according to Chinese patents (CN 108795964, published on 13.11.2018; CN 116949019, published on 27.10.2023) is the lack of a protease cleavage site for removing the affinity tag; the presented genetic constructs do not allow cleaving off affinity tags for X-ray structural studies of picornaine 3C of rhinovirus A28.

A disadvantage of the prototype [2] is the presence of a TEV protease cleavage site for cleaving the affinity tag. TEV protease has a number of limitations: due to the high value of the Michaelis constant K _M (50-90 μM) [7], for a high rate of enzymatic reaction (150 substrate molecules per hour), the substrate concentration should exceed 100-200 μM, at a temperature of 25°C. To achieve complete proteolytic cleavage of the target product, it is necessary to either add a large amount of enzyme or incubate for 3-16 hours at a temperature of 16-30°C. SENP1 protease at a temperature of 0°C exhibits highly efficient enzymatic activity (7-14 thousand substrate molecules per hour), which weakly depends on the substrate/protease ratio [8]. Thus, the cleavage of the SUMO affinity tag from the recombinant 3C A28 protease will occur more efficiently, and no amino acid residues of the linker regions of the hydrolysis site will remain at the site of hydrolysis, as in the case of TEV.

In this regard, there is a need to develop genetic constructs and producers of chimeric picornaine 3C of rhinovirus A28, allowing to obtain the 3C enzyme not only for protein engineering purposes, but also for the purpose of studying the properties of the 3C protease of the poorly studied rhinovirus serotype A28. The presence of the affinity tag 14xHis-SUMO increases the solubility of the recombinant chimeric protein while maintaining enzymatic activity, while the tag can be effectively cleaved by SENP1 protease to obtain a viral protease with an authentic amino acid sequence.

Disclosure of invention

The technical result of the claimed invention is the production of a recombinant protein 3C (picornaine 3C of rhinovirus A28) for the cleavage of fusion proteins and the cleavage of affinity tags, as well as for studying the properties of viral protease 3C A28 with an authentic amino acid sequence.

The specified technical result is achieved by obtaining recombinant plasmid DNA pB2k_T5_SUMO_HRV_3C, which ensures the synthesis of the chimeric protein SUMO-HRV_3C in E.coli cells, has a size of 4193 bp and contains the following elements of the genetic structure in accordance with the physical and genetic map presented in Fig. 1:

- a nucleotide sequence encoding the leading affinity tag of histidines 14xHis tag, located in the SUMO open reading frame, having coordinates from 778 to 858 bp;

- a gene encoding a small ubiquitin-like protein SUMO, having coordinates from 868 to 1101 bp and included in the sequence SEQ ID NO: 2, encoding the chimeric protein SUMO-URV 3C;

- the picornaine 3C gene of rhinovirus A28, with a nucleotide sequence having coordinates from 1105 to 1653 bp and included in the sequence SEQ ID NO: 2, encoding the chimeric protein SUMO-HRV3 C;

- the T5 bacteriophage promoter PN25/04 (T5 promoter), overlapping with the lac operator of the lactose operon, having coordinates from 672 to 717 bp;

- a nucleotide sequence encoding the SET1 peptide used as a label to increase solubility, having coordinates from 1666 to 1717 bp;

- bacteriophage T7 transcription terminator, with coordinates from 1721 to 1728 bp;

- the aminoglycoside 3'-phosphotransferase gene KanR, used as a genetic marker that provides resistance of E.coli transformant cells to first-generation aminoglycoside antibiotics, with coordinates from 3057 to 3872 bp;

- unique restriction sites Xhol, EcoRI, with coordinates from 1660 to 1665 and from 751 to 756 bp, respectively;

- origin of replication of plasmids p15A ori, having coordinates from 1 to 648 bp."

The specified technical result is also achieved by creating the KRX/pB2k_T5_SUMO_HRV_3C strain, producing the SUMO-HRV_3C chimeric protein obtained by transforming E. coli cells of the KRX strain with the recombinant plasmid DNA pB2k_T5_SUMO_HRV_3C according to item 1, with a calculated molecular weight of 32.5 kDa.

The significant differences between the claimed invention and the prototype, ensuring the achievement of the technical result, are:

1. Use of the genetic sequence of the rhinovirus isolate of serotype A28, which has no more than 52% homology with the prototype serotype B14.

2. The use of SUMO as a partner protein, which, according to published data [5], is more effective than the MBP affinity label in increasing the solubility of chimeric proteins.

3. The possibility of cleaving synthetic affinity tags from the authentic amino acid sequence of the viral protease 3C A28 using the SUMO protease SENP1 instead of the TEV protease, which increases the efficiency of hydrolysis at a temperature of 4°C and also eliminates the presence of amino acid residues of the linker regions of the protease recognition sites (which is typical for the TEV site).

The essence of the claimed invention is explained by drawings.

Fig. 1 shows the physical and genetic map of the recombinant plasmid DNA pB2k_T5_SUMO_HRV_3C.

Fig. 2 shows a scheme for obtaining recombinant plasmid DNA pB2k_T5_SUMO_HRV_3C, which ensures the synthesis of the chimeric protein SUMO-HRV 3C in E. coli cells.

Fig. 3 shows the expected picture of the electropherogram of the recombinant plasmid DNA pB2k_T5_SUMO_HRV_3C after hydrolysis with restriction endonucleases EcoRI and XhoI (1), EcoRV (2), AvaII (3), NsiI (4).

Figure 4 shows a fragment of the chromatogram of sequencing of plasmid DNA pB2k_T5_SUMO_HRV_3C, demonstrating the absence of a frameshift between the SUMO and 3C sequences, as well as the absence of heterologous amino acid residues after the SUMO protease recognition site.

Fig. 5 shows an electropherogram of the separation of protein fractions in SDS PAGE after the first (1, 2) and second (3) chromatography of recombinant picornaine 3C, which shows: the chimeric protein SUMO-HRV_3C before the addition of SENP1 protease (1); the products of enzymatic cleavage of the SUMO fusion partner (2); the fraction of proteins not bound to Ni-NTA Sepharose (3).

Fig. 6 shows an SDS PAGE electropherogram showing: (1) recombinant domain 1 of Yezo virus nucleoprotein (d1 N Yezo virus) after cleavage of the affinity tag with recombinant picornain 3C of rhinovirus A28 and subsequent affinity reverse chromatography (fraction of d1 N Yezo virus protein not bound to Ni-NTA Sepharose after application) (14.2 kDa); (2) chromatographic peak during affinity reverse chromatography after proteolysis of d1 N Yezo virus with recombinant picornain 3C of rhinovirus A28 (fraction of chimeric d1 N Yezo virus protein bound to Ni-NTA Sepharose after application); (3) chimeric d1 N Yezo virus protein before affinity chromatography (cell lysate); (4) chimeric protein d1 N Yezo virus after the first affinity chromatography before enzymatic cleavage of the affinity tag with recombinant picornain 3C of rhinovirus A28 (18.9 kDa).

Fig. 7 shows the nucleotide sequence SEQ ID NO:1 of the recombinant plasmid DNA pB2k_T5_SUMO_HRV_3C.

Fig. 8 shows the nucleotide sequence SEQ ID NO:2 encoding the chimeric protein SUMO-HRV_3C. Fig. 9 shows the amino acid sequence SEQ ID NO:3 of the recombinant protein 3C A28.

Implementation of the invention

Example 1. Construction of the plasmid genetic construct pB2k_T5_SUMO_HRV_3C, intended for expression of the chimeric gene SUMO-HRV_3C.

The DNA fragment encoding the rhinovirus A28 protease 3C was obtained by PCR amplification from a cDNA copy of the rhinovirus A28 isolate genome containing the full-length picornaine 3C gene. For this purpose, primers (Table 1) flanking the 3C protease gene and introducing BamHI and XhoI restriction endonucleases recognition sites before and after the gene, respectively, were used. The DNA fragment encoding the 14-His tag and the SUMO partner protein gene was also amplified by PCR using primers introducing NdeI (before the 14-His tag) and BamHI (after the SUMO gene) restriction endonucleases recognition sites. The PCR products were purified by electrophoretic separation in agarose gel, after which the amplicons were cloned in a targeted manner into the expression plasmid vector pB2k_T5_mNG at the recognition sites of restriction endonucleases NdeI and XhoI by replacing the mNG gene (Fig. 2).

Table 1 - Oligonucleotide primers used in the work

Primer name Nucleotide sequence 5'-3' F-HRV-3C-BamHI GTG GTT GGA TCC GGA CCG CAG GAG GAA TTT GGG AGA R-HRV_3C_XhoI TTT TGA CTC GAG CTA TTA TTG TTG TTC AGT GAA ATA TGA CCT 1CP_NdeI AGATATCATATGAGCAAACACCATC 16CP CGG ATC CAC CTG TCT GAT GCA GCA TTG CAT CAA TTT CGT C

The obtained genetic construct was used to transform electrocompetent cells of the E. coli strain NEBStable (New England Biolabs, USA) with subsequent cultivation on LB agar medium containing the selective antibiotic kanamycin (50 μg/ml) at 30°C for 16-18 hours. Single colonies were then selected and cultured in liquid LB nutrient medium containing 50 μg/ml kanamycin at 30°C for 12-16 hours. Plasmid DNA of the transformant clones was extracted using the Monarch Plasmid Miniprep Kit (New England Biolabs, USA). The correctness of the nucleotide sequence of the plasmid DNA of the transformant clones was confirmed by restriction analysis at the EcoRI, XhoI hydrolysis sites (Fig. 3) and subsequent sequencing using the Sanger method (Fig. 4).

Example 2. Obtaining a producer strain by transforming E.coli cells of the recipient strain KRX with the plasmid construct pB2k_T5_SUMO_HRV_3C.

The strain producing the chimeric protein SUMO-HRV_3C was obtained by electroporation of competent cells of the E. coli strain KRX (Promega, USA) followed by cultivation on SOB agar medium containing 50 μg/ml kanamycin at 37°C for 16-18 hours. Single colonies were subcultured into liquid SOB medium containing 50 μg/ml kanamycin and incubated in an orbital shaker at 37°C and 220 rpm for 8-10 hours until OD ₆₀₀ ~ 1 was reached.

Example 3. Biosynthesis of the chimeric protein SUMO-HRV_3C by the method of induced cultivation of the producer strain E. coli KRX/pB2k_T5_SUMO_HRV_3C.

Induction of expression of the chimeric protein SUMO-HRV_3C was carried out by adding isopropyl-β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, followed by cultivation for 20-24 hours at 18°C and 220 rpm.

Example 4. Isolation, cleavage of the partner protein and chromatographic purification of recombinant picornaine 3C from E. coli KRX/pB2k_T5_SUMO_HRV_3C cells.

The bacterial biomass was precipitated by centrifugation at 4500 g for 15 min at 4 °C, the cell pellet was resuspended in buffer A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.0) in a ratio of 15 ml of buffer per 1 gram of cell pellet. The resuspended cells were disrupted under pressure in an EmulsiFlex-C3 homogenizer (Avestin, Canada), after which the lysate was centrifuged at 13000 g for 30 min at 4 °C to remove intact cells and cellular debris, the supernatant was passed through a filter with a pore width of 0.22 μm. Isolation of recombinant proteins from the supernatant was performed by metal chelate affinity chromatography. The resulting supernatant was applied to the Ni-NTA-Sepharose column sorbent (GE Healthcare, UK), which has a high binding capacity for His-tagged recombinant proteins. After application, the column was washed with 5 column volumes of buffer A with a stepwise increase in imidazole concentration: 50, 100 and 150 mM. The target proteins were eluted with buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 8.0). The chromatographically purified SUMO-HRV_3C protein was dialyzed against buffer A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.0). The resulting chromatographic protein retains enzymatic activity and can be used in the procedures of hydrolysis of chimeric proteins containing the recognition site of 3C protease.

The affinity tag 14xHis-SUMO can affect the biochemical properties of the enzyme and can also become an obstacle for structural studies. In this regard, to obtain picornaine with the native amino acid sequence, the chimeric protein SUMO-HRV_3C was treated with SUMO protease SENP1, available in the laboratory, in a mass ratio of protease to protein of 1:1000, at a temperature of 4 °C for 4-16 h to cleave the affinity tag 14-His_SUMO from the recombinant picornaine 3C. The hydrolysis products were separated by reverse chromatography using metal-chelate affinity sorbents. The protein fraction was applied to a column containing Ni-NTA-sepharose, on which 14-His-SUMO, SENP1 protease and ballast proteins were bound to the stationary phase, and the target protein without 3C affinity tags remained in the mobile fraction, which passed through the sorbent, since native picornain 3C A28 has no affinity for the sorbent.

Protein fractions collected during purification were separated in 12% SDS PAGE under denaturing conditions according to the Laemmli method. 4x loading buffer (50 mM Tris-HCl, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) was added to 30 μl of protein solution (~10 μg) and incubated for 5 min at 98°C. Then the samples were loaded into the wells of 12% gel and electrophoresis was performed for 30 min at 60 V in a stacking gel and 60 min at 180 V in a resolving gel. Then the gel was placed in a staining solution (1.5% Coomassie R250, 10% acetic acid, 20% ethanol) for 40 min with constant stirring, then washed several times with water. Figure 5 shows the results of SDS PAGE, where the fraction of the chimeric protein SUMO-HRV_3C before the addition of SENP1 protease (1), the products of enzymatic cleavage of the affinity label SUMO (2) and the fraction of proteins that did not bind to Ni-NTA-Sepharose after application during flow chromatography, in particular picornaine 3C (3) are shown. It is shown that picornaine 3C corresponds to the theoretically calculated mass of 20 kDa and also has a high degree of purity and homogeneity.

Example 5. Confirmation of the enzymatic activity of recombinant picornaine 3C using the example of hydrolysis of a chimeric protein containing the recombinant domain 1 of the Yezo virus nucleoprotein (d1 N Yezo virus).

The enzymatic activity of recombinant picornaine 3C was confirmed using the chimeric recombinant protein 6xHis-3C-d1N_Yezo_virus, which is the Yezo virus nucleoprotein domain 1 and the 6xHis-3C fusion peptide for subsequent proteolytic cleavage of the 6xHis affinity tag from the target gene by picornaine 3C. 3C protease was mixed with 1 mg of 6xHis-3C-d1N_Yezo_virus substrate and incubated at 4°C for 16 h. The products of enzymatic cleavage of the 6xHis-3C affinity tag from the d1N_Yezo_virus target protein were separated on 12% SDS PAGE under denaturing conditions according to the Laemmli method as described in point 4. Analysis of protein fractions by SDS PAGE (Fig. 6) showed that chromatographically purified 3C has a strictly specific proteolytic activity directed at the Leu-Gln-Ala-Ile-Phe-Gln↓Gly-Pro recognition and cleavage site and is suitable for biotechnological, biochemical and molecular biological applications where highly specific hydrolysis of amino acid sequences is required.

Thus, a construct and producer strain were obtained for the production and purification of recombinant picornaine 3C of rhinovirus A28, which has high purity and exhibits strictly specific proteolytic activity with respect to a substrate containing a cleavage site for 3C protease.

Sources of scientific, technical and patent information

1. Tishin A.E., Gladysheva A.V., Pyatavina L.A., Olkin S.E., Gladysheva A.A., Imatdinov I.R., Vlaskina A.V., Nikolaeva A.Yu., Samygina V.R., Agafonov A.P. Preparation and crystallization of recombinant picornaine 3C of rhinovirus A28. // Crystallography. - 2023. - T. 68. - No. 6. - P. 926-933.

2. Blom N. et al. Cleavage site analysis in picornaviral polyproteins: discovering cellular targets by neural networks // Protein Science. - 1996. - T. 5. - No. 11. - pp. 2203-2216. (prototype)

3. Ullah R. et al. Activity of the human rhinovirus 3C protease studied in various buffers, additives and detergents solutions for recombinant protein production // PloS one. - 2016. - T. 11. - No. 4. - P. e0153436.

4. Yong Jingui, Du Pan, Lisen. Recombinate production method of the PRESCISSION enzymes in Escherichia coli // Chinese Patent CN 108795964, 11/13/2018.

5. Zhang Xu, Dong Lu, Lu Pan. Preparation method of high-yield recombinant HRV-3C enzyme // Chinese Patent CN 116949019, 10/27/2023. https://blast.ncbi.nlm.nih.gov

6. Marblestone J. G. et al. Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO // Protein science. - 2006. - T. 15. - No. 1. - pp. 182-189.

7. Nam H. et al. Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self-cleavage exhibits enhanced enzymatic activity // FEBS Open bio. - 2020. - T. 10. - No. 4. - pp. 619-626.

8. Frey S., Görlich D. A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins // Journal of chromatography A. - 2014. - T. 1337. - P. 95-105.

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tgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaac

agtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagc

aaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaata

tctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaa

caaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgc

tgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacga

cgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctgggg

caaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccg

tctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctctccccgcgcgttggccga

ttcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagctaaggaagctaaaatg

agccatattcaacgggaaacgtcttgctccaggccgcgattaaattccaacatggatgctgatttatatg

ggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccga

tgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagg

ctaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcat

ggttactcaccactgcgatcccagggaaaacagcattccaggtattagaagaatatcctgattcaggtga

aaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtcctttt

aacggcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttggtgcgagtg

attttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccatt

ctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaatta

ataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaact

gcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatat

gaataaattgcagtttcacttgatgctcgatgagtttttctaataaggtcaaataaaacgaaaggctcag

tcgaaagactgggcctttcgttttaatctgctagcggagtgtatactggcttactatgttggcactgatg

agggtgtcagtgaagtgcttcatgtggcaggagaaaaaaggctgcaccggtgcgtcagcagaatatgtga

tacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaa

tggcttacgaacggggcggagattcctggaagatgccaggaagatacttaacagggaagtgagaggggcc

gcggcaaagccgtt</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>789</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..789</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q4">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gcacatatcaatctgaaagttaaaggtcaggatggcaacgaagtgtttt

tccgtattaaacgtagcacccagctgaaaaagctgatgaatgcatattgtgatcgtcagagcgttgatat

gaccgcaattgcatttctgtttgatggtcgtcgtctgcgtgcagaacagacaccggatgaactggaaatg

gaagatggtgacgaaattgatgcaatgctgcatcagacaggtggatccggaccgcaggaggaatttggga

gaagcttaattaaacataacacttgtgttgtaacaacaagcaatgggaaattcaccgggttaggaatata

tgataatgttatgatcatcccaacacatgctgatgcaggcaaggaagtggaaatagatggaatcaaaact

caagttgatgatgcctatgacctgcataactcccaaggaattaaattagaaattactgtcctcaagttgc

acagaaatgaaaaattcagagatatcagaaaatacataccagagtctgaagatgactacccagagtgtca

tttagcgctagtcgctaaccaacaagaacccaccatactagaagttggtgactgctgctcatatggaaat

atactcttgagtggtaatcaaacagctaggatgattaagtacaattacccaacaaaatcaggattttgtg

gtggtgttttatacaaaatagggttagttttaggcatacatgttgggggaaatggtagggatggattttc

tgcaatgttattaaggtcatatttcactgaacaacaataa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="3">

<INSDSeq>

<INSDSeq_length>183</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..183</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q6">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>GPQEEFGRSLIKHNTCVVTTSNGKFTGLGIYDNVMIIPTHADAGKEVEI

DGIKTQVDDAYDLHNSQGIKLEITVLKLHRNEKFRDIRKYIPESEDDYPECHLALVANQQEPTILEVGDC

CSYGNILLSGNQTARMIKYNYPTKSGFCGGVLYKIGLVLGIHVGGNGRDGFSAMLLRSYFTEQQ</INSD

Seq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (11)

1. Recombinant plasmid DNA pB2k_T5_SUMO_HRV_3C, providing the synthesis of the chimeric protein SUMO-HRV_3C in E. coli cells, having the nucleotide sequence SEQ ID NO: 1, 4193 bp in size and containing the following elements in accordance with the physical and genetic map: a nucleotide sequence encoding the leading affinity tag of histidines 14xHis tag, located in the SUMO open reading frame, having coordinates from 778 to 858 bp; a gene encoding a small ubiquitin-like protein SUMO, having coordinates from 868 to 1101 bp and included in the sequence SEQ ID NO: 2, encoding the chimeric protein SUMO-HRV_3C; the picornaine 3C gene of rhinovirus A28, with a nucleotide sequence having coordinates from 1105 to 1653 bp and included in the sequence SEQ ID NO: 2, encoding the chimeric protein SUMO-HRV_3C; the T5 bacteriophage promoter PN25/04 (T5 promoter), overlapping with the lac operator of the lactose operon, having coordinates from 672 to 717 bp; a nucleotide sequence encoding the SET1 peptide used as a label to enhance solubility, having coordinates from 1666 to 1717 bp; bacteriophage T7 transcription terminator with coordinates from 1721 to 1728 bp; the aminoglycoside 3'-phosphotransferase gene KanR, used as a genetic marker providing resistance of E. coli transformant cells to first-generation aminoglycoside antibiotics, with coordinates from 3057 to 3872 bp; unique restriction sites XhoI, EcoRI, with coordinates from 1660 to 1665 and from 751 to 756 bp, respectively; origin of replication of plasmids p15A ori, having coordinates from 1 to 648 bp. 2. A recombinant producer strain of E. coli KRX/pB2k_T5_SUMO_HRV_3C obtained by transforming E. coli cells of the recipient strain KRX with the recombinant plasmid DNA pB2k_T5_SUMO_HRV_3C according to item 1 and ensuring the synthesis of the chimeric protein SUMO-HRV_3C with a calculated molecular weight of 32.5 kDa.