RU2482185C2 - Method for preparing and expressing coding sequence of isoform 2 of human renalase gene - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to genetic engineering and may be used in biotechnology to produce proteins of practical interest, which are a product of silent genes or low expression genes. What is presented is a method for producing a recombinant protein of human renalase 2 involving a) preparing a complete cDNA sequence, and b) expressing the prepared sequence in E.coli, wherein the stage (a) is performed by PCR amplification of each exon using specific primer pairs and a genomic DNA as a matrix, followed by twinning requiring no pre-treatment and involving the same method for adjacent exons at first, and then being the product of their amplicon combining. In this case, the primers twice exceeding exons in number are selected so that the forward primer contain a terminal sequence of the preceding exon and an initial sequence of the exon to be copied; the downstream primers contain only a sequence complementary to the end of the amplified exons, while the forward and downstream primers flanking the complete coding sequence contain restriction sites necessary for integration into a plasmid vector.

EFFECT: preparing the proteins of practical interest which are the products of silent genes or low expression genes.

2 tbl, 7 dwg, 1 ex

Description

The invention relates to the field of molecular biology, molecular genetics, biotechnology and medicine, and in particular to a method for obtaining the complete coding sequence of a human renalase 2 gene with a DNA template. This method is based on the production of a method of polymerase chain reaction (PCR) with a DNA matrix of individual coding sequences of a gene (exons), which are then combined by PCR into a full-length coding sequence (cDNA) using complementary cDNA terminal fragments. Using this method, two transcriptional forms of the human renalase gene were obtained: renalase 2 cDNA and renalase 1 cDNA used as a control, and their expression was performed in the prokaryotic system.

The structure and features of expression of the human renalase gene

The human renalase gene (NC_000010.10) is located on the 10th chromosome of the q23.33 locus and includes 309469 base pairs and has 10 exons. According to computer analysis, the renalase gene exists in at least two splicing variants - transcription variants of mRNA 1 and 2. Transcriptional variant 1 (renalase 1 mRNA, MN_001031709.2) encodes a protein in 342 amino acid residues (a.o.) and includes 7 exons: 1-4, 6, 7 and 9 exons (NP_001026879) with an estimated molecular weight of 37.85 kDa and pI of 6.06. Renalase 1 was discovered in 2005 in silico and can serve as a clear illustration of the effective use of postgenomic technologies (see review [1]).

Expression of the renalase 1 gene in human tissues is mainly found in the glomeruli and proximal tubules of the kidneys, as well as in cardiomyocytes, liver and skeletal muscle [2].

Other authors [3] showed that the expression of renalase 1 is also determined in the peripheral nerves, adrenal glands, and human adipose tissue.

The second isoform (mRNA of renalase 2, MN_0018363.3) also contains 7 exons (1-4, 6, 7 and 10) and differs from renalase 1 only in the last exon. According to computer prediction, renalase 2 encodes a protein of 315 aa. (NP_060833) with an estimated molecular weight of 34.95 kDa and a pI of 6.27. However, so far this protein has not been detected in human tissues [2]. True, using the reverse transcription PCR method (RT_PCR), it was possible to detect mRNA of renalase 2 in the adrenal gland and hypothalamus [3]. Using the same method, another form of the renalase gene isoform, the so-called renalase 3, the mRNA of which contains exons 1, 4, 6, 7 and 9 and encodes a theoretically calculated amino acid sequence of 232 a.a. (AK296262) [3]. In principle, a fourth variant of splicing of the renalase gene (renalase 4 mRNA) containing 5, 6, 7, 8, and 9 exons that encode a theoretically calculated amino acid sequence of 138 a.o. [four].

However, with the exception of renalase 1, protein products encoded by renalase mRNAs 2, 3, and 4 have not yet been detected, and therefore their biological role is unknown.

Traditionally, the cDNA cloning method requires the isolation and purification of a significant amount of poly-A selective mRNA from a specific tissue or cells (tissue / cells in which there is an expression cloned gene). It is especially difficult to isolate the necessary mRNA with a low content in the cell, and in the case of human tissues this is also associated with compliance with legally established procedures.

The discovery of Taq polymerase [5-6] made it possible to develop a polymerase chain reaction (PCR) method, with the advent of which it became possible to amplify the gene of interest directly from the isolated mRNA, which greatly simplified the method of gene cloning. Despite this, there still remains the need to isolate specific mRNA from tissues.

With the development of the PCR method technology and the chemical synthesis of oligonucleotides, recombinant genes are obtained directly from cDNA libraries, flanking DNA sections containing restriction endonuclease sites necessary for cloning into the corresponding vectors are added to the coding regions [7-8].

To obtain cDNA of renalase genes, we used the method of obtaining the coding sequence of a gene directly from genomic DNA, which does not require isolation and purification of mRNA and therefore significantly simplifies and speeds up this procedure.

The prototype of this method of obtaining cDNA and expression of human renalases 1 and 2 in the literature is absent. Similar approaches were used in [9–12]. So, in [9], a method was described for obtaining the complete coding sequence (cDNA) of the human PIGR gene (polymeric immunoglobulin receptor), which includes 10 exons, by combining single exons obtained from the DNA matrix by PCR (Genomic DNA Splicing). Initially, the authors obtained individual exons in separate reaction media using direct and reverse primers optimized for computer analysis for each exon. The second round of amplification was carried out after preliminary purification of the PCR product. In the second round of amplification, individual exons were synthesized using primers containing nucleotide sequences overlapping neighboring exons. At the final stage, exons were combined by PCR, thus obtaining a full-length cDNA sequence. Davies et al. [10] proposed a method for obtaining the full-length cDNA sequence of the SWS1 gene containing 5 exons from total lemur DNA. And in this case, individual exons were obtained by PCR in separate reaction media from a DNA template using direct and reverse primers containing nucleotide sequences that overlap neighboring exons. After purification of the PCR product, exons were combined in pairs to obtain a full-length cDNA sequence. Flanking primers of the cDNA sequence included restriction sites for insertion into the expression vector.

Booth et al. [12] amplified individual exons of the human ciliary neurotrophic factor (CNTF) containing only two exons and one intron. At the beginning, each of the exons was individually amplified by PCR, and then these exons were combined and inserted into a plasmid vector to obtain the complete CNTF coding sequence. In this case, the technology of the so-called ligation-independent cloning (ligation-independent cloning - without the use of restriction endonucleases and ligase) was used, developed previously [13-14]. It is based on the use of oligonucleotide primers in which at the 5'-end the deoxythymidine residues are replaced by deoxyurocyl residues. After PCR, the amplicons were treated with uracil DNA glycosylase (UDG), which generates 3 ′ protruding “sticky” ends in amplicons. At these ends, there is a non-covalent association of the PCR fragments and their covalent incorporation into the expression vector. However, the use of such a technology of combining three or more exons to obtain the complete coding sequence of a gene is not known in the modern literature, and methodological features are not described.

Recombinant PCR methods are widely used in the cloning of various genes and the creation of hybrid genes. To combine the two DNA fragments by PCR, the first PCR fragment is obtained at the beginning, in which a DNA fragment (about 20 nucleotides) complementary to the second 3'-terminal DNA fragment is completed at the 5'-end using a synthetic oligonucleotide. After annealing the first PCR fragment and the second DNA fragment, PCR amplification of the common DNA fragment with end primers for the common fragment is performed [15-17].

Recently, projects related to sequencing of the genomes of various eukaryotes have been implemented - this is the Human Genome project, aimed at determining the nucleotide sequence of the entire human genome [18-19], as well as projects to determine the complete nucleotide sequence of species genomes. New DNA sequencing approaches are being developed that allow the sequencing of various types of genomes quickly and with minimal cost. Analysis of genomic sequences using certain algorithms (gene prediction method) allows one to determine genomic structures with sufficient accuracy. The presence of complete genomic sequences and easy accessibility to obtain amplificates of any DNA fragments from genomic DNA and the combination of PCR fragments make it possible to construct any genetic constructs with a known nucleotide sequence.

The development of chemically automatic synthesis of oligonucleotides led to an increase in synthesis from 30 to several tens, made it possible to extend the oligonucleotide matrix by PCR to several hundred nucleotides. The strategy of stepwise growth of the nucleotide sequence as a result of several PCRs allowed the synthesis of a gene 400 bp long (bp) [20]. In other studies, it was possible to carry out chemical synthesis of a gene on a synthetic matrix with a length of more than 650 bp using two and one nucleotide chain as a matrix for PCR [21-23]. However, chemical gene synthesis is currently a laborious and expensive work, and is not widely used to obtain complete coding regions of genes.

Disclosure of invention

In the available scientific literature, we did not find any work on obtaining cDNA of the unexpressed renalase 2 gene by direct synthesis of structural units (exons) with a genomic DNA matrix, followed by combining these structural units using the PCR method. We used the exon method to clone a functionally weakly expressed or silent renalase 2 gene. This method is based on the production of a recombinant gene from the total DNA of an organism from its known exon structures. This method allows you to quickly "collect" a gene from a bank of the total number of genes with known nucleotide sequences of the eukaryotic genome. To obtain an unexpressed renalase 2 gene, we first worked out this technique on the expressed renalase 1 gene. According to the nucleotide sequence of the data bank (GenBank www.ncbi. Homo sapiens chromosome [gi: 51511726]) locus NC_000010 gene "C10orf59" has two transcriptional variants: the first is the locus NM_001031709 1477 bp mRNA (renalase gene 1); the second option is the locus NM_018363 2107 bp mRNA (renalase 2 gene). The human renalase gene 1 includes 7 exons and contains 342 aa NP_001026879 (hypothetical protein LOC55328 isoform 1). The renalase 2 gene also includes 7 exons, differs from transcriptional variant 1 by the seventh exon and contains 315 aa NP_060833.1 (hypothetical protein LOC55328 isoform 2). As a result, expression plasmid vectors pET-Ren-I and pET-Ren-II were constructed that produced the recombinant form of renalase 1 and renalase 2 in Escherichia coli cells of the Rosetta strain.

The algorithm for designing primers for the synthesis of the complete coding region of any gene is universal. In the nucleotide sequence data bank (GenBank www.ncbi.), The sequence of the desired gene is selected. Based on the example of the renalase gene, the NC_000010 locus, according to automatic computational analysis using the gene prediction method, includes two transcriptional mRNAs (NM_001031709 and NM_018363). The first transcript (renalase 1) includes seven coding regions (exons) with nucleotide numbers 62-179, 945-1050, 1543-1685, 10191-10349, 220527-220700, 268611-268786, 297746-297898. Therefore, for the synthesis of the complete coding sequence of renalase 1 by PCR, it is necessary to synthesize 14 primers (seven “direct” and seven “reverse” primers).

Direct primers (Re1-for - forward) are constructed in two parts. The first part consists (from the 3'-end) of the sequence (20 ± 3 nucleotides) of the end of the previous exon, i.e. for renalase 1, the nucleotide numbers, starting from the second exon, are: 159-179, 1030-1050, 1666-1685, 10331-10349, 220679-220700, 268766-268786 of the NC_000010 locus. Variation in the length of the nucleotide sequence depends on the AT and GC composition. This part of the primer serves as a link between exons (linker region). The first part of the direct primer of the first exon (the direct flanking region of the complete structural gene - 1Re1-for) can contain up to 15 nucleotides, serves to be inserted into the plasmid vector and contains the restriction site necessary for this. In table 1, the first part of the primer is shown in italics and bold. The second part also consists of 20 ± 3 nucleotides, including the beginning of exons, i.e. direct nucleotide numbers starting from the first exon: 62-79, 945-962, 1543-1563 10191-10209, 220527-220547, 268631-268648 and 927746-927762 of the NC_000010 locus.

From the above it follows that, for example, the direct primer of the second exon (2Re1-for) consists of twenty-one (159-179) and eighteen (945-962) nucleotides of the NC_000010 locus (see table 1).

Reverse primers (Re1-rev - reverse) are constructed from one part (20 ± 3 nucleotides) of the complementary sequence, which consists of the end part of the exon, i.e. numbers from the 5'-end of the complementary sequence, starting from the first exon: 159-179, 1187-1050, 1666-1685, 10333-10349, 220679-220700, 268764-268786, 297878-297898 of the locus NC_000010. The reverse primer of the last exon (the reverse flanking region of the complete structural gene 7Re1-rev) contains an additional portion of the nucleotide sequence (up to 15 nucleotides), which is used for insertion into the plasmid vector and contains the necessary restriction site (see table 1).

Thus, the structure of the primers can be created automatically based on the known nucleotide sequence of the gene according to automatic computational analysis using the gene prediction method.

The synthesis of the structural gene for human renalase 1 was carried out in several stages (see Fig. 1).

At the first stage, total DNA was obtained from human whole blood lymphocytes using the modified method [16]. DNA was isolated in 2 ml Eppendorf tubes. To 1 ml of blood containing EDTA was added 1 ml of buffer-1 (22% sucrose; 20 mM MgCl ₂ ; 20 mM Tris-HCl pH 7.5; 1% Triton X-100). The solution was centrifuged for 10 min at 3800 rpm on an Eppendorf Centrifuge 5415 R microcentrifuge. The pellet was resuspended in 1 ml of buffer-1 (diluted twice) and centrifugation was repeated. The cells were then suspended in 0.9 ml of buffer-2 (10 mM Tris-HCl; 2 mM EDTA; 400 mM NaCl), 0.1 ml of 10% SDS and proteinase K were added to a concentration of 1 mg / ml. The solution was incubated for 2-3 hours at 58 ° C with periodic stirring. Then, twice phenol-chloroform extraction was carried out, after which 0.1 volumes of 3 M sodium asetate pH 5.0 and 0.6 volumes of isopropanol were added. DNA was besieged at 12,000 rpm (using the same centrifuge) and the pellet was washed with 70% cold ethanol. DNA was dissolved with 0.2 ml of H ₂ O and stored at -20 ° C.

At the second stage, seven single exons were obtained by PCR on a human DNA matrix. Each exon was synthesized separately using a pair of primers: for the first exon, 1Re1-for n + 62-79 and 1Re1-rev 159-179; the second exon 2Re1-for 159-179 + 945-962 and 2Re1-rev 1187-1050; the third exon 3Re1-for 1030-1050 + 1543-1563 and 3Re1-rev 1666-1685; the fourth exon 4Re1-for 1666-1685 + 10191-10213 and 4Re1-rev 10333-10349; fifth exon 5Re1-for 10333-10349 + 220527-220547 and 5Re1-rev 220679-220700; the sixth exon 6Re1-for 220679-220700 + 268631-268648 and 6Re1-rev 268764-267886; seventh exon 7Re1-for 268766-268786 + 927746-927762 and 7Re1-rev 297878-297898 + n (see table 1 - from 1 to 14 primers). Direct primers (Re1-for ...) contain at the 3'-end a sequence of approximately 20 ± 3 nucleotides that is complementary to the reverse primer of the previous exon (Re1-rev ...) - the linker region of PCR fragments, which is the link between exons (at Fig. 1, the linker region is indicated by the symbol L). In table 1, this area is shown in italics and bold.

For example, the linker region of the forward primer 2Re1-for 159-179 + 945-962 is complementary to the reverse primer 1Re1-rev 159-179. In Figure 1, this region was designated as L2 (L (n) - linker, n - exon number) - linker region, a link between exons. The linker regions of flanking exons 1R1 and 7R1 of the renalase gene - direct primer 1Re1-for n + 62-79 exon 1R1 and reverse primer 7Re1-rev 297878-297898 + n exon 7R1, contain the restriction enzyme sites BamHI and XhoI, respectively (Table 1 - No. 1 and 14, underlined regions), which are used for subsequent insertion of the structural renalase gene into the pET-28a (+) plasmid vector. Reverse primers (Re1-rev - reverse) begin at the exon boundary and their nucleotide sequence is included in the structure of this exon.

PCR was carried out in 20 μl of a reaction mixture containing: 4 μl of a 5x PCR solution (33.5 mM Tris pH 8.8; 8.3 mM (NH ₄ ) ₂ SO ₄ ; 0.1% Twin 20; 5 mM MgCl ₂ ; 0 , 5 mM dNTP; 5% glycerol), primers (400 μM each in a given volume), 50-100 ng of DNA and 0.5 units. Tag DNA polymerase (modified for thermal start). PCR conditions: 95 ° C - 5 min 1 cycle; 92 ° C - 20 sec, 55 ° C - 15 sec, 72 ° C - 10 sec 35 cycles; 72 ° C - final cycle 3 min.

In the synthesis of single exons, minor bands are usually formed with a good yield (1, 2, 4, and 6 exons). However, the 3rd and 7th exons did not give a visible band of the PCR product or gave a low yield of the PCR product (5th exon). In this case, at the first stage of PCR, the DNA concentration of the template was increased by a factor of 2–3 or reamplification was performed. Reamplification was performed under the same conditions as the initial PCR. The exception was that instead of the DNA template, amplicons from the first PCR mixture were used in an amount of 1-2 μl, and the number of cycles was reduced to 30.

As a result of seven separate PCR, amplicons of the following exons were obtained: 1Re1, 2Re1, 3Re1, 4Re1, 5Re1, 6Re1, 7Re1 (Fig. 1 - stage 2, Fig. 2, track numbers 1 to 7).

At the third stage, PCR was used to pair exons 1Re1 with 2Re1, 3Re1 with 4Re1, and 5Re1 with 6Re1. Linker L2 served as a link for exons 1Re1 and 2Re1, linker L4 for exons 3Re1 and 4Re1, and linker L6 for 5Re1 and 6Re1. The amplicons of two single exons served as a matrix, and 1Re1-for n + 62-79 and 2Re1-rev 1187-1050 served as primers for the first exon association; for the second association - 3Re1-for 1030-1050 + 1543-1563 and 4Re1-rev 10333-10349; the third association - 5Re1-for 10333-10349 + 220527-220547 and 6Re1-rev 268764-268786. PCR was performed under the same conditions as for single exons. As a matrix, 0.5-1 μl of the PCR mixture from amplicons of single exons was used. When combining the two exons, an insignificant admixture of primers and genomic DNA located in amplicons of single exons did not interfere with the synthesis of paired exons and did not produce by-products.

As a result of the synthesis of three separate PCR, three DNA fragments were obtained for two combined exons: 1Re1-2Re1, 3Re1-4Re1, 5Re1-6Re1 (Fig. 1 - stage 3, Fig. 3 - lanes 1, 2, 3).

At the fourth stage, two double exons were combined by PCR by the method: 1Re1-2Re1 with 3Re1-4Re1 and 5Re1-6Re1 with 7Re1. Linker L3 served to link exons 1Re1-2Re1 and 3Re1-4Re1, the amplicons of these exons were used as a matrix, and 1Re1-for n + 62-79 and 4Re1-rev 10333-10349 were used as primers. Linker L7 bound the double combined exon 5Re1-6Re1 and single exon 7Re1, the amplicons of these exons were used as a matrix, and 5Re1-for 10333-10349 + 220527-220547 and 7Re1-rev 297878-297898 + n were used as primers.

PCR conditions were similar to those used for double combining of exons. The matrix was 0.5-1 μl of the PCR mixture from double exon amplicons. When exons were combined by PCR, an insignificant admixture of primers located in the amplicons of two combined exons did not interfere with the combination of four and three exons and did not produce a by-product.

As a result of synthesis by PCR, four combined exons 1Re1-4Re1 and three combined exons 5Re1-7Re1 were obtained (Fig. 1 - the 4th stage, Fig. 3 - lanes 4, 5 and see table 2).

At the fifth stage, the complete coding region of the renalase 1 gene was synthesized, combining all seven exons by PCR. For this, four combined exons 1Re1-4Re1 were combined with three combined exons 5Re1-7Re1. The linker for combining these exons was the L5 linker, the amplicons of these exons were used as the matrix, and 1Re1-for n + 62-79 and 7Re1-rev 297878-297898 + n were used as primers.

PCR conditions were similar to those used in the fifth stage of the renalase gene synthesis. As a matrix, 0.5-1 μl of the PCR mixture from amplicons of the combined exons was used. When combining PCR exons by the method, an insignificant admixture of primers and amplicons in the PCR mixture from the fourth stage of gene synthesis did not interfere with the synthesis of the entire coding region of the gene.

As a result of PCR, the complete renalase 1 gene was obtained, consisting of seven exons (1Re1-7Re1), the flanking ends of which contained L1 and L8 linkers with restriction sites for BamHI and XhoI, respectively (Fig. 1 - 5th stage, Fig. 3 - lane 6 and table 2).

The combined DIC fragment was amplified, purified electrophoretically and isolated from 2% agarose gel. For this, 10 μl of a solution from the PCR reaction was applied to a well of an agarose gel. After electrophoresis, the necessary band was also cut out from the gel, and DNA was eluted according to the recipe and using the Wizard SV Gel and PCR Clean-Up System (Promega) kit.

To test the integrity of the coding sequence of the structural renalase 1 gene, the combined DNA fragment was inserted into the plasmid vector pGEM-T vector designed for cloning the PCR product. This vector contains a protruding single T-residue at the 3'-ends, which greatly enhances the ligation of the PCR product. After cloning the combined PCR product, 10 clones were selected, which were first subjected to restriction analysis and then sequencing of the PCR fragment.

DNA sequencing was performed on a capillary DNA sequencer (Applied Biosystems 310 DNA analyzer) using the BigDye Terminator v 3.1 Cycle Sequencing Kit in accordance with the recommendations of the manufacturer. The obtained DNA sequence was correlated with the renalase 1 gene (locus BC005364 GeneBank) using the BLAST program, available on the website of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).

As a result of sequencing of three clones, it was found that in the cloned sequence there are three point mutations at positions 107, 342, and 723 (in Fig. 3 they are indicated with a capital letter, in bold, and italics with underline). These mutations affect the third nucleotide of degenerate triplets and therefore do not change the amino acids encoded by these triplets.

It was previously shown that PCR can introduce mutations in amplified DNA, which are the result of insufficiently high replicative accuracy of Taq DNA polymerase. To reduce mutational pressure, the total number of cycles in DNA amplification is reduced, or thermostable DNA polymerase with correcting activity is used [21-23]. For our purposes, we used thermostable DNA polymerase (Eurogen company), which is a specially developed mixture of thermostable DNA polymerases for the efficient amplification of long DNA fragments and possesses corrective 3 '-> 5' exonuclease activity.

At the sixth stage, the expression vector of the recombinant renalase gene 1 pET-Ren-I was obtained by insertion into the plasmid vector pET-28a (f +) at the BamHI and XhoI restriction sites of a PCR DNA fragment consisting of seven exons joined together. To obtain this PCR expression vector, a product consisting of the complete coding region of the renalase 1 gene (fifth step Scheme 1) was treated with restriction enzymes BamHI and XhoI and ligated with the prepared linear vector pET-28a (f +) flanked by the same restriction enzyme recognition sites. As a result, the expression vector pET-Ren-I was obtained (see Fig. 1), which transformed E. coli cells.

Rubidium cells of E. coli K12 (strain DE3 Rosetta) were prepared according to the modified Kushner method [27], aliquots of competent cells (0.2 ml each) were stored at -70 ° C. Transformation was carried out according to the standard Cohen method [28] using 0.1 μg of ligation products. Transformed cells were plated on plates with LB agar containing 100 μg / ml ampicillin (Am). After incubation at 37 ° C overnight, an individual colony grown on a plate was grown in 4 ml of LB medium with 100 μg / ml Am overnight. The overnight culture was subcultured in 200 ml of the same LB medium and the cell culture was increased to an optical density of 0.7-0.8 units. Then, IPTG was added to a concentration of 1 mM and the cells continued to incubate with vigorous stirring for another 3 hours. Biomass was collected by centrifugation and stored at -20 ° C.

The presence of renalase 1 gene expression was analyzed electrophoretically. The cell sample was boiled in the application buffer (60 mM Tris-HCl pH 6.8; 1% SDS; 0.2 mM EDTA; 5% mercaptoethanol; 8% glycerol; 001% bromphenol blue) and analyzed in 0.1% SDS- 10% PAGE. Protein was detected by staining in Coomassie diamond blue R-250 (see Fig. 5). According to the calculated data, the renalase 1 protein contains 342 amino acid residues (amino acid residues) and corresponds to the calculated mass of 37.83 kDa and pI of 6.06.

Obtaining the complete coding sequence of renalase 2

Transcriptional variant 1 (renalase 1), as well as transcriptional variant 2 (renalase 2), was first detected by the gene prediction method [2], however, renalase 2 has not yet been detected in human cells.

The transcriptional variant of mRNA 2 differs from the transcriptional variant of mRNA 1 by the seventh exon. In addition, in the first exon, the coding amino acid sequence of renalase 2 contains at position 37 the remainder of glutamic acid (codon - GAC), and renalase 1 - aspartic acid (codon - GAG). The codon of this amino acid is included in the primers 1Re2-rev and 2Re2-for (in Fig. 4 and Table 1, the position of this a.k. is in bold letters in capital letters (cf. 2, 2-1 and 3, 3-1) Taking into account the replacement of the amino acid in the first exon and the synthesis of the new seventh exon, four additional primers were synthesized (see table 1, numbers: 2-1, 3-1, 13-1, 14-1). The complete coding sequence of renalase 2 was synthesized according to the same scheme, differences in the synthesis were manifested starting from the second stage (see Fig. 1).

At the second stage, three single exons were obtained by PCR using the PCR matrix: 1Re2, 2Re2 and 7Re2 (see table 2, numbers 1-1, 2-1 and 7-1). Single exons were synthesized using a pair of primers: for 1Re2 exon, primers 1Re1-for n + 62-79 and 1Re2-rev 159-179; for exon 2Re2, primers 2Re2-for 159-179 + 945-962 and 2Re1-rev 1187-1050; for the seventh exon, primers 7Re2-for 268766-268786 + 308220-308240 and 7Re2-rev 308271-308291 + n (see table 1 and 2).

At the third stage, only paired association of 1Re2 exons with 2Re2 was performed by PCR. To combine these exons, 1Re1-for n + 62-79 and 2Re1-rev 1187-1050 served as primers. As a result of PCR, the DNA fragment 1Re2-2Re2 was obtained (see table 1 and 2).

At the fourth stage, double exons were combined by PCR by the method: 1Re2-2Re2 with 3Re1-4Re1 and 5Re1-6Re1 with 7Re2. For combining to the quaternary exon 1Re2-4Re2, the primers were 1Re1-for n + 62-79 and 4Re1-rev 10333-10349, and for the tertiary 5Re2-7Re2, the primers were 5Re1-for 10331-10349 + 220527-220547 and 7Re2-rev 308271- 308291 + n (see Fig. 1 - the 4th stage and table 1 and 2).

At the fifth stage, a recombinant renalase 2 gene was obtained, combining all seven exons by PCR. As a result of the combined four exons 1Re2-4Re2 with three exons 5Re2-7Re2, the complete coding sequence of renalase 2 1Re2-7Re2 was obtained. For the synthesis of this sequence, 1Re1-for n + 62-79 and 7Re2-rev 308271-308291 + n served as primers (see Fig. 1 - the 5th stage and table 1 and 2).

At the sixth step, the expression vector of the recombinant renalase 2 gene was constructed. To obtain the PCR expression vector, the product from step 6 was treated with restriction enzymes BamHI and XhoI and ligated with the prepared linear vector pET-28a (f +) flanked by the same restriction enzyme recognition sites. As a result, the expression vector pET-Ren-II was obtained (see Fig. 1-6, stage), which transformed E. coli cells. As a result of expression, a renalase 2 protein was obtained that contains 315 aa and corresponds to an estimated mass of 34.95 kDa and a pI of 6.27 (see Fig. 6).

Summarizing the results obtained using the exon method of recombinant protein cloning that we propose, the following advantages of this method should be noted: 1 - this method is a fairly simple method of cloning recombinant genes that does not require significant time and material costs compared to the generally accepted at present; 2 - for the synthesis of the gene coding sequence, primers are selected automatically based on the data bank of the nucleotide sequence of the cloned gene, the number of primers is equal to twice the number of exons of the cloned gene; 3 - this method does not require the isolation of mRNA from biological objects; 4 - using this method, the recombinant renalase 2 protein gene was cloned, which refers to proteins with a low level of expression or to a silent gene; 5 - this method does not require cells and tissues of animals in which the expression of the cloned gene is increased, hard-to-reach human tissues are difficult to obtain, the preparation of which can be coupled (for example, in case of post-mortem material) in compliance with legally established procedures; 6 - using this method (when setting it in a stream mode), a significant amount of recombinant genes can be synthesized in a short period of time.

The technical result of the invention is the production of recombinant genes - the gene for renalase 1, consisting of 342 a.a., and the gene for renalase 2, consisting of 315 a.a., and their expression in bacterial cells (Figs. 5 and 6).

Brief description of tables and figures

Table 1. PCR primers to obtain the complete coding sequence of the renalase genes 1 and 2.

Note. In the structure of primers, in italics and in bold, the overlapping region (L - linker) of the (n + 1) th exon with the nth exon is indicated, i.e., the region of the 2Re1-for primer 159-179 + 945-964 is complementary to the primer 1Re1 -rev 159-179. The flanking linker regions of the first exon of the direct primer of the first exon (1Re1-for n + 62-79) and the reverse primer of the seventh exon (7Re1-rev 297878-297898 + n) contain the restriction sites BamHI and XhoI, respectively - nucleotides are indicated in bold and italics and are indicated the character "n". Primers numbered 2-1, 3-1, 13-1, 14-1 are synthesized to obtain the coding sequence of the renalase gene 2. See the text for an explanation.

Table 2. The sizes of exons of the renalase genes 1 and 2, obtained as a result of PCR.

Notes. Re1 and Re2 denote exons specific for the renalase genes 1 and 2, respectively, and the numbers in front of these symbols are the exon sequence number.

Figure 1. Exon method for producing recombinant human renalase 1.

Figure 2. Electrophoresis of PCR DNA fragments of single exons in a 2% agarose gel. Led - DNA markers ranging in size from 50 bp up to 500 bp with an interval of 50 bp .; 1 - exon 1Re 131 bp; 2 - exon 2Re 126 bp; 3 - exon 3Re 166 bp .; 4 - exon 4Re 179 bp .; 5 - exon 5Re 193 bp .; 6 - exon 6Re 199 bp .; 7 - exon 7Re 188 bp The sizes of exons are given taking into account the size of the linker (see Table 2 and Scheme I). Arrows indicate fragments of DNA marker.

Figure 3. Electrophoresis of PCR fragments of DICs of combined exons in a 2% agarose gel. Led1 - DNA markers ranging in size from 50 bp - 500 bp with an interval of 50 bp .; Led2 - 50 bp DNA markers and from 100 bp - 1000 bp with an interval of 100 bp .; 1 - exons 1Re-2Re 236 bp; 2 - exons 3Re-4Re 325 bp; 3 - exons 5Re-6Re 370 bp .; 4 - exons 1Re-4Re 538 bp .; 5 - exons 5Re-7Re 535 bp .; 6 - exons 1Re-7Re 1061 bp The sizes of exons are given taking into account the size of the linker (see Table 2 and Fig. 1). Arrows indicate fragments of DNA marker.

Figure 4. Comparison of the nucleotide sequences of the renalase gene — transcript 1 from the NCBI GenBank (access code NM 001031709) (A) and the nucleotide sequence of the renalase gene we cloned (transcript 1) (B).

Figure 5. Expression of renalase 1 in E. coli cells. 1 - total cellular protein without induction; 2 - total cellular protein after IPTG induction; 3 - protein molecular weight marker (Fermentas SM0441) - 19, 25, 34, 49, 85, and 117 kDa.

Figure 6. Expression of renalase 1 and 2 in E. coli cells. 2 and 5 - total cellular protein without induction; 1 - total cellular protein after induction of IPTG (renalase 1); 3 and 4 - total cellular protein after induction of IPTG (renalase 2); 6 - protein renalase 1 after purification on Ni-agarose; 7 - protein molecular weight marker (Fermentas SM0441) - 19, 25, 34, 49, 85, and 117 kDa.

This work was supported by the Russian Foundation for Basic Research (grant No. 11-04-01181-a).

Claims (1)

A method for the preparation and expression of a complete cDNA sequence of human renalase isoform 2, which comprises the steps of:
(1) - obtaining total human DNA containing the target gene;
(2) - PCR amplification in a separate reaction volume of each of the exons using the following primer pairs, respectively:
IRe1-for n + 62-79, 1Re2-rev 159-179;
2Re2-for 159-179 + 945-962, 2Re1-rev 1187-1050;
3Re1-for 1030-1050 + 1543-1563, 3Re1-rev 1666-1685;
4Re1-for 1666-1685 + 10191-10213, 4Re1-rev 10333-10349;
5Re1-for 10333-10349 + 220527-220547, 5Re1-rev 220679-220700;
6Re1-for 220679-220700 + 268631-268648, 6Re1-rev 268764-268786;
7Re2-for 268766-268786 + 308220-308240, 7Re2-rev 308271-308291 + n,
where the given range of values corresponds to the positions of the nucleotides in the renalase gene NC_000010;
(3) - obtaining a full-sized cDNA by pairwise combining without preliminary purification first of the neighboring exons obtained in stage (2), and then being the products of their combination of amplicons using the corresponding forward and reverse primers selected from the above list according to table 2;
(4) - construction of a recombinant plasmid vector suitable for use in E. coli and containing the full-length cDNA sequence obtained in the previous step;
(5) - transformation of the E. coli strain with the specified vector;
(6) - cultivation of the transformed strain under conditions ensuring the biosynthesis of human renalase isoform 2 protein in bacterial cells in the form of inclusion bodies.

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