RU2614124C9 - Optimized gene encoding recombinant ipfiii protein - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nucleotide sequence encoding a recombinant interferon lambda protein is optimized for expression in E. coli cells and cloned into the rET30a plasmid. E. coli BL-30-L cell, intended for interferon lambda recombinant protein production is obtained by transformation of E. coli BL (DE3) cells by rET30a plasmid containing the said nucleic acid. The invention provides a recombinant interferon lambda protein with biological activity of 2.1*109 U/mcmol in MDBK/VSV system.

EFFECT: increased biological activity.

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Description

The invention relates to the field of biotechnology, in particular to the production of genetic constructs containing an optimized nucleotide sequence that encodes a recombinant protein IPFIII.

Viral hepatitis C (HCV) is a major cause of chronic liver diseases such as cirrhosis and hepatocytic carcinoma. HCV is one of the main problems of world health, about 3% of the world's population suffer from chronic HCV. Currently, HCV therapy is based on the use of type I interferons, primarily interferons alpha 2a and 2b. However, the treatment efficiency does not reach 55%, when using high doses of interferon alfa, side effects are often manifested, which leads to a decrease in the quality of life of patients and causes a forced interruption of treatment.

One of the methods for improving the treatment of HCV can be the use of other variants of interferon alpha molecules with increased antiviral activity.

Interferons (IFN) are a group of highly homologous proteins produced by various types of cells in response to a viral infection. All IFN interact equally effectively with their specific receptor, which triggers a cascade of reactions that ultimately lead to the activation of a large number of interferon-sensitive genes mediating the specific biological properties of IFN. Although all IFNs have different antiviral properties, only IFN-alpha 2a / b has historically been used for anti-HCV therapy. Recent studies have revealed types of IFN that are less toxic than IFN-alpha, in particular IFN-lambda. Moreover, IFN-lambda has a very high anti-HCV activity profile.

The natural gene encoding human IFN-lambda is not adapted for efficient expression in E. coli. This is due to the presence of AGG and AGA codons rarely found in E. coli for arginine, as well as to the presence of sequences homologous to the regulatory regions of E. coli genes, which leads to interruption of translation or translation initiation from internal regions of the gene (SD sites). The combination of these causes leads to low expression efficiency (not more than 5%), which does not allow developing a commercially viable technology for the preparation of preparations based on IFN-lambda.

The presence of codons suboptimal for E. coli is characteristic of all genes encoding interferons. Optimized genes encoding target proteins are used to overcome this problem. Patent RU No. 2354703 describes an optimized gene sequence encoding IL-21 (prototype). The amino acid codons are used based on the frequency of occurrence, for example, the AGC codon was used for serine, even for the starting amino acid. The experience of the applicant indicates that for serine, it is necessary to take the TST (usually) or TCC codons. The first ten codons are extremely important for efficient expression and the applicant proposes a different nucleotide composition of the starting site.

However, for the treatment of HCV, interferon preparations are administered parenterally. Due to the short half-life, interferons must be administered every second day at high doses. High doses cause numerous side effects and lead to the formation of autoantibodies. These antibodies reduce the effectiveness, or even force to interrupt the course of treatment. The overall percentage of withdrawal from IFN therapy for HCV is extremely high. It was necessary to develop methods that increase the lifetime of interferon in the cytoplasm. This is achievable by increasing the physical volume (hydrodynamic radius) to slow down the filtration processes from blood plasma through the pores of the renal tubules. Molecular enlargement is possible by creating nucleotide sequences encoding interferon and a stabilizing molecule. Such molecules can be long-lived human blood proteins or artificial polypeptide molecules, usually consisting of charged amino acids and serine residues with glycine.

Therefore, the objective of the invention was the development of a recombinant interferon lambda protein for the treatment of HCV, which will overcome the disadvantages of existing interferon alpha drugs.

Applicant has developed and prepared a nucleic acid encoding a recombinant interferon lambda protein represented by SEQ ID NO: 2 and characterized by a nucleotide sequence with SEQ ID NO: 1. As well as an E. coli BL-30-L Cell for the production of a recombinant lambda interferon protein with SEQ ID NO: 2 obtained by transforming E. coli BL (DE3) cells with plasmid pET30a containing the nucleic acid of claim 1.

The nucleotide sequence was cloned into plasmid pET30a, resulting in the recombinant plasmid p30-L. Recombinant plasmid p30-L was used to transform E. coli cells of strain BL21 (DE3), and a stable producer strain of E. coli BL-30-L was obtained.

The technical result of the claimed invention is that the obtained genetic construct, the nucleic acid represented by SEQ ID NO: 1, allows efficient synthesis of the recombinant interferon lambda protein with SEQ ID NO: 2 with antiviral activity in an amount of more than 30% in E. coli cells from total cellular protein.

Brief Description of the Drawings

FIG. 1. Electrophoresis of E. coli BL21 (DE3) clones transformed with p30-L plasmid after induction. The arrow indicates the target product - recombinant human lambda. Track 1 - corresponds to the producer strain BL-30-L.

FIG. 2. Chromatographic profile of the passage of the recombinant lambda protein through SP-Sepharose. The major peak corresponds to the yield of the recombinant lambda protein.

FIG. 3. Chromatographic profile of elution of the recombinant lambda protein from the Kromasil 300-5C18 analytical column.

FIG. 4. Electrophoresis of cell lysates by fermentation. Tracks 1 - molecular weight standards, tracks 2-9 correspond to fermentations 1-8. The arrow indicates the position of the recombinant interferon lambda protein in the gel.

THE INVENTION EXAMPLES EXAMPLES

EXAMPLE 1. OPTIMIZATION OF A NUCLEOTIDE SEQUENCE

The codons of the nucleotide sequence encoding the recombinant lambda protein have been optimized for expression in E. coli cells by substituting codons often found in the E. coli K12 genome. The frequency of codon usage in the E. coli genome is taken from: Y Nakamura, K Wada, Y Wada, H Doi, S Kanaya, T Gojobori, and T Ikemura. Codon usage tabulated from the international DNA sequence databases. Nucleic Acids Res. 1996 Jan 1; 24 (1): 214-215.

Additionally, part of the CTG codons encoding leucine was replaced by TTA or TTG codons, all AGC codons encoding serine were replaced by TTC or TCC codons. Substitutions were introduced in order to eliminate internal SD sites. The developed optimized nucleotide sequence is represented by SEQ ID NO: 1.

EXAMPLE 2. OBTAINING PLASMID P30-L

The optimized nucleotide sequence encoding the recombinant lambda protein was hydrolyzed by the restriction enzymes NdeI and HindIII. The commercial plasmid pET30a was also hydrolyzed with the restriction enzymes NdeI and HindIII. The hydrolysis products were separated by electrophoresis in 1% agarose, the products were isolated from the gel, combined and ligated with T4 DNA ligase. XL-1 cells were transformed with a ligase mixture, plated on a Petri dish and incubated for 16 hours at 37 ° C. The grown clones were analyzed for the presence of the target plasmid by PCR. Clones containing the recombinant plasmid were grown, and the target product, plasmid p30-L, was isolated from the obtained biomass.

EXAMPLE 3. PRODUCTION STRAIN PRODUCTION

Plasmid p30-L transformed E. coli cells of strain BL21 (DE3), seeded on a Petri dish and incubated for 16 hours at 37 ° C. The grown clones were analyzed for expression of the target protein by electrophoresis in SDS page. Clones expressing the highest amount of protein (Fig. 1) were selected as the producer strain BL-30-L. Cells of producer strains were grown and stored at -70 ° C.

EXAMPLE 4. ISOLATION OF RECOMBINANT PROTEIN

The cells of the producer strain were subcultured into a culture flask with 200 ml LB containing 25 mg / ml kanamycin. The culture was grown on a shaker at 280 rpm at 37 ° C until the optical density reached OD585 = 0.8E. Protein expression was induced by the addition of IPTG to a concentration of 1 mM. The culture was additionally cultured for 4 hours, then the cells were precipitated by centrifugation at 5000 g. Cell pellet was lysed in 8M urea and processed on an ultrasonic disintegrator. The cell lysate was centrifuged at 20,000 g for 2 hours. The supernatant was filtered through a Q-Sepharose column. The fraction that did not bind to this sorbent was chromatographed on SP-Sepharose in a NaCl gradient (start buffer: 20 mM TrisHCl pH 6.8, 20 mM NaCl, final buffer 20 mM TrisHCl pH 6.8, 400 mM NaCl). The chromatographic profile is shown in FIG. 2. Fractions containing the desired product were combined and dialyzed in 10 mM acetic acid. The purity of the product was analyzed by HPLC (Fig. 3). The biological activity of the isolated recombinant interferon lambda protein in the MDBK / VSV system was 2.1 * 10 ⁹ U / μmol. The sequence of the recombinant lambda interferon protein is represented by SEQ ID NO: 2.

EXAMPLE 5. Analysis of the presence of a recombinant protein interferon lambda in cell cultures

According to the results, the optical density of the culture medium and the mass of bacterial cells after centrifugation were measured. The results are shown in table 1.

The presence and amount of the recombinant lambda interferon protein was determined by electrophoresis in SDS page. Figure 13 shows the gels after electrophoresis of all fermentations. Applied cells located in 10 μl of culture. In FIG. 4 shows the homogeneity of the protein spectrum in all fermentations, except fermentation No. 4 (track No. 5). The biomass of this fermentation was not used for further work. Fermentation biomasses 1-3 and 5-8 (tracks 2-4 and 6-9) were combined. According to an estimate of the total amount of the recombinant lambda interferon protein, we can conclude that its mass (before chromatographic purification and refolding) is 2.23 g (9% by weight of dried biomass according to electrophoresis). A recombinant protein of at least 95% electrophoretic purity is obtained. The expression efficiency of the recombinant protein with SEQ ID NO: 2 was more than 30%.

EXAMPLE 6. Methodology for assessing the antiviral activity of the recombinant protein interferon lambda

The antiviral activity of the recombinant lambda interferon protein was evaluated using the vesicular stomatitis virus (VSV) strain indiana. VSV is a member of the family Rhabdoviridae of the genus Vesiculovirus and has an 11 kb RNA gene of negative polarity that encodes five structural proteins: nucleocapsid (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and large polymerase protein (L). There are two serotypes of VSV New Jersey (VSV-NJ) and Indiana (VSV-I). There are some clinical differences between serotypes; New Jersey generally produces more severe clinical changes and has a shorter incubation period. Therefore, VSV-I is a convenient and safe model for studying the antiviral properties of various drugs.

To assess the antiviral properties of the obtained recombinant protein, MDBK cells (bovine fetal kidney cells) were seeded in a 96-well plate in an amount of 1 × 104 cells / well in 100 μl of complete DMEM medium (Gibco), followed by incubation at 37 ° C in 5% CO ₂ atmosphere until a monolayer is formed (approximately 1 day). The complete DMEM medium was replaced with 100 μl of DMEM support medium containing the studied preparations at various concentrations (eight binary dilutions).

Eight binary dilutions of commercial IFN-I (from 1: 1 × 105 to 1: 128 × 10 ⁵ ) and commercial preparation of human IL-29 (R&D) were used as a positive control. As a negative control, appropriate concentrations of human serum albumin (HSA) were used, as well as DMEM support medium without the addition of any drugs. Next, cells with the studied drugs and control samples were incubated at 37 ° C in 5% CO ₂ atmosphere for 1 day. Then, 100 LD50 of VSV virus was added to each well, followed by incubation at 37 ° C in 5% CO ₂ atmosphere for 1 day until the cytopathic effect appeared. The cytopathic effect of VSV was evaluated using light microscopy.

The protective effect of the drugs was evaluated in comparison with the control samples: IFN-I, IL-29, which have proven antiviral effects. The antiviral activity of the drugs (EC50) was expressed as the concentration of the drug, providing a 50% protective effect and was calculated according to the Spearman-Kerber formula:

where EC50 is the concentration of the drug, which has a 50% antiviral effect;

Dmax is the binary logarithm of dilution, above which 100% protection has occurred;

d is the binary logarithm of the dilution step (1 in the case of binary titration);

n is the number of holes for each dose of the drug (4);

p is the number of wells that provided protection in Dmax and in subsequent dilutions.

Antiviral activity of preparations of the recombinant protein interferon lambda

Initially, 73 samples were screened and the protective concentration of EC50 was calculated using the MDBK / VSV model. As it turned out, 19 of 73 drugs had a pronounced antiviral activity (marked in gray in table 2). EC50 values for the recombinant lambda interferon protein ranged from 1.5 to less than 0.4 ng / ml, which is significantly larger than the commercial comparison drug IL-29.

After the initial screening of 73 obtained samples from 19 active variants, 14 were selected for a more detailed study. Additional experiments were conducted and the EC50 was calculated for each of the variants (table 3). For most drugs, with the exception of “7-4,” the EC50 values were in the range of ≈ 500 pg / ml, while for the comparison drug IL-29 EC50 = 66.5 ng / ml. Thus, we can conclude that the obtained recombinant lambda protein with SEQ ID NO: 2 in the MDBK / VSV model is not inferior in its antiviral activity to similar commercial comparison drugs.

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Sequence listing

<110> FSBI "SSC Institute of Immunology" FMBA of Russia

<120> "OPTIMIZED GENE CODING RECOMBINANT PROTEIN IPFIII"

<160> NUMBER OF SEQ ID NOS: 2

<210> SEQ ID NO 1

<211> 1126

<212> DNA

<213> artificial

<400> SEQUENCE 1:

CATATGTGTGATCTGCCGCAGACCCATTCTCTGGGTAACCGTCGTGCACTGATCCTCTTGGCTCAGAT GCGCGTAAAACAAATTGAATCTAAAACTAAATTCCAAGAAGCATTAGATGCTGCAGGTGATAAACTGG TGGTTGTGGATTTCTCTGCAACCTGGTGCGGTCCGTGCAAGATGATCAAACCGTTCTTTCACTCTGCA TCTGAGAAATACTCCAACGTGATCTTCCTGGAAGATGTTGACGATTGCCAGGATGTTGCATCTGAGTG CGAAGTGAAATGCATGCCGACCTTCCAGTTCTTCAAGAAAGGTCAGAAAGTTGGCGAGTTCTCTGGTG CTAACAAAGAGAAGCTGGAAGCGACCATCAACGAACTGGTTGGTGGTTCTGGCGGTTCCAAAGGTGGC GGTTCCGGTGGTGGTAAATCTAAAGGCGGTGGCAGCGGTGGCTTGAAACTCCTGAAGTCCAAAGGTAG CTCTGGCGGCAGCGGTAAAGGTTCCGGCAAAGGTTCCAAAGGTGGTGGTTCCCACCACCACCATCACC ACGGTAGCGGCGGCTCTGGTTCCGGTAGCGGATCCCCAGTTCCGACTTCTAAACCGACTACCACTGGC AAAGGCTGCCACATCGGTCGTTTCAAATCTCTGTCTCCACAAGAACTGGCATCTTTCAAGAAAGCTCG TGATGCACTGGAAGAGTCTCTGAAACTGAAAAACTGGTCTTGTTCCTCTCCAGTGTTCCCAGGCAACT GGGATCTGCGTCTCTTGCAGGTTCGTGAACGTCCGGTGGCACTGGAAGCTGAGTTGGCACTGACTCTC AAAGTGCTGGAGGCTGCAGCTGGTCCAGCACTGGAAGATGTGCTCGATCAGCCGCTGCATACTCTGCA CCATATCCTGTCTCAGCTCCAAGCTTGCATCCAGCCGCAACCAACTGCAGGTCCACGTCCGCGCGGTC GTCTGCACCATTGGTTGCATCGTCTGCAGGAAGCTCCGAAGAAAGAGTCTGCAGGCTGCTTGGAAGCA TCTGTGACCTTCAACCTGTTTCGTCTGCTGACTCGTGATCTCAAATACGTGGCAGATGGTAACCTGTG CTTGCGCACCTCTACTCATCCAGAGTCTACTTAAGCTT

<210> SEQ ID NO 2

<211> 372

<212> AA (amino acids)

<213> artificial

<400> SEQUENCE 2:

MCDLPQTHSLGNRRALILLAQMRVKQIESKTKFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSAS EKYSNVIFLEDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELVGGSGGSKGGG SGGGKSKGGGSGGLKLLKSKGSSGGSGKGSGKGSKGGGSHHHHHHGSGGSGSGSGSPVPTSKPTTTGK GCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLK VLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEAS VTFNLFRLLTRDLKYVADGNLCLRTSTHPEST

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Claims (2)

1. A nucleic acid encoding a recombinant lambda interferon protein represented by SEQ ID NO: 2 and characterized by the nucleotide sequence of SEQ ID NO: 1. 2. An E. coli BL-30-L cell for the production of a recombinant interferon lambda protein with SEQ ID NO: 2 obtained by transforming E. coli BL (DE3) cells with plasmid pET30a containing the nucleic acid of claim 1.

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