RU2221868C2 - Gene encoding l-asparaginase in erwinia carotovora and strain escherichia coli vkpm = b-8174 as producer of erwinia caratovora l-asparaginase - Google Patents

Abstract

FIELD: biotechnology, genetic engineering, enzymology, biochemistry. SUBSTANCE: DNA fragment encoding the complete amino acid sequence of L-asparaginase from Erwinia caratovora (ECAR-LANS) is isolated and characterized. Plasmid DNA pBADLANS is constructed wherein enzyme-encoding DNA fragment is under control of regulated promotor pBAD. As result of transformation of E. coli cells with proposed recombinant plasmid and the following selection of transformed clones the new strain is prepared: producer ECAR-LANS (E. coli VKPM = B-8174) providing high yield of recombinant enzyme. The main designation of this enzyme is it using as the main component in preparing the known and development of new antitumor preparations. EFFECT: improved preparing method, valuable medicinal properties of enzyme. 2 cl, 7 dwg, 4 tbl, 4 ex

Description

 The invention relates to biotechnology and, in particular, to genetic engineering, and is a DNA fragment encoding the complete amino acid sequence of Erwinia carotovora L asparaginase, and pBADLANS recombinant plasmid DNA, which causes the synthesis of Erwinia carotovora L asparaginase (ECAR-LANS) in Escherichia coli cells .

 The invention may find application in the microbiological industry and medicine, in particular for the creation of new antitumor agents.

 Bacterial asparaginases are well-known enzyme preparations that have been used in antitumor therapy for the treatment of acute lymphoblastic leukemia for more than 30 years / 1 /. The effectiveness of the use of asparaginases in antitumor therapy is limited by the development of the immune response to the administered drugs / 2 /. In this regard, for the necessary courses of combined chemo- and enzyme therapy of leukemia, it is advisable to have a set of L-asparaginases that have high enzymatic activity, but differ in their antigenic properties. Additional opportunities for targeted changes in the enzymatic and immunological properties of L-asparginases are opened using genetic engineering methods / 3 /. These circumstances determine the interest in the cloning of new genes for bacterial L-asparaginases and their modification through site-directed mutagenesis and heterologous expression.

 To date, the primary structures of the genes of bacterial Erwinia chryzantemi / 4 /, Escherichia coli / 5 /, Wolinella succinogenes / 6 / and a number of other microorganisms are known, and recombinant plasmid DNAs providing the synthesis of L-asparaginases in Escherichia coli / 7 / bacteria cells have been described. and Erwinia carotovora / 8 /. The primary structure of the Erwinia carotovora L-asparaginase gene was not previously known, nor was the preparation of recombinant strains causing the synthesis of this enzyme described.

 The aim of the invention is the cloning of the ECAR-LANS gene and the preparation of a recombinant strain of bacteria producing biologically active ECAR-LANS in high yield. This goal is achieved by the fact that the DNA fragment of the Erwinia carotovora strain that encodes the complete amino acid sequence of the ECAR-LANS gene was isolated, as well as by the fact that the recombinant plasmid DNA pBADLANS was designed to ensure the synthesis of ECAR-LANS in E. coli cells.

 The essence of the modified ECAR-LANS gene is that it has the nucleotide sequence shown in figure 1, which encodes the synthesis of ECAR-LANS protein with the amino acid sequence indicated in the same figure.

The essence of the recombinant plasmid pBADLANS, whose restriction and genetic map is presented in figure 2, is that it contains a fragment of the natural gene ECAR-LANS, integrated into the expression yeast vector pBAD24. Plasmid pBADLANS consists of the following elements:
a 4.5 kb EcoRI fragment of plasmid pBAD24 / 9 /,
- 1.1 kb EcoRI fragment carrying the ECAR-LANS gene.

 The molecular weight of plasmid pBADLANS is 3.5 Md, which corresponds to a size of 5.6 TPN.

 The number of copies of the obtained plasmid is 50-100 per E. coli cell.

 Plasmid pBADLANS is replicated in E. coli cells due to the replicon pUC18. The synthesis of ECAR-LANS in cells in plasmid pBADLANS transformed with E. coli strains is carried out under the control of the regulated pBAD promoter and the rrnBTIT Escherichia coli terminator and is achieved by culturing the transformed strain on ordinary selective media by adding L-arabinose to a concentration of 0.2%.

Plasmid PBADLANS contains the genes:
- A modified natural ECAR-LANS gene that encodes the synthesis of this protein in a bacterial cell. This gene, introduced at the EcoRI cleavage site, has the nucleotide sequence shown in FIG. This sequence encodes a full-sized precursor ECAR-LANS with its own signal peptide for export of protein into the periplasm and subsequent processing;
- bla-gene, providing synthesis of b-lactamase and affecting the resistance of E. coli strains carrying the plasmid to ampicillin;
- araC gene, which is a regulator of the pBAD promoter.

The producer strain ECAR-LANS is obtained by transformation of Escherichia coli cells BL21 (DE3) / 10 / [F-, ompT, hsdS _B (r _in- , m _in- ), dcm, gal, λ (DE3), Amp ^R ] of the proposed recombinant plasmid DNA. The choice of strain is due to the fact that it carries, as well as the fact that it is defective in the synthesis of proteases, which significantly affects the yield of synthesized heterologous proteins.

The transformed BL21 / pBADLANS clone obtained after selection is capable of producing ECAR-LANS upon cultivation. The level of synthesis of ECAR-LANS in the constructed strain is mg / L with a culture titer of 1 × 10 ⁹ cells / ml, which follows from the data of the determination of ECAR-LANS in biomass samples of the producer strain using a specific enzymatic reaction.

 The resulting bacterial strain carrying the plasmid PBADLANS is characterized by the following general characteristics.

 Morphological signs: Cells have an oblong, palatoid shape, do not bud during division.

 Cultural traits: Cells grow well on commonly used culture media. The generation time is about 30 minutes in a liquid LB medium. On 2-2.5% nutrient agar "Difco" round, smooth, yellowish colonies with smooth edges are formed. When grown on liquid LB and YT media, intense even turbidity forms.

Physiological and biochemical characteristics: The optimum cultivation temperature is from 35 to 37 ^o C, the optimum pH is 7.6.

 The source of nitrogen is organic compounds (in the form of tryptone, yeast extract).

 The strain is deposited in the All-Russian collection of industrial microorganisms under the number VKPM.

 The method for cloning the ECAR-LANS gene and producing the recombinant plasmid pBADLANS is illustrated by the following examples.

Example 1. Isolation of the central fragment of the ECAR-LANS gene
For cloning the ECAR-LANS gene, a strategy based on the method of inverted polymerase chain reaction "(" inverted PCR, 1) and schematically shown in figure 3 was used.

 At the first stage, the amino acid and nucleotide sequences of bacterial L-asparaginases were compared using information available on the Internet at the Sanger Center website in the UK / 11 /, as well as at the website of the US National Center for Biotechnological Information / 12 /. Using the CLUSTAL X / 13 / program, the most conserved regions of L-asparaginases of bacterial origin were identified (Fig. 4) and two synthetic oligonucleotides Mot3 and Mot4 were synthesized, the structure and properties of which are shown in Table 1. These oligonucleotides were used as primers in PCR with genomic DNA samples isolated in a conventional manner / 14 / from various strains of nervinia found in the laboratory collection. PCR was performed using Taq-DNA polymerase manufactured by Fermentas (Vilnius) in accordance with the manufacturer's instructions on an MJ-Research instrument under the following conditions: initial heating - 2 min, 94, then 25 amplification cycles - 94, 40 ". 50, 40 " 72, 40 ". The volume of the reaction mixture was 50 μl, the concentration of primers was 0.5 μm, the DNA concentration was 20 ng / μl. In the DNA samples isolated from strains of nervinia, conventionally designated 1A, 2A, 204, a reaction product with a size of about The 300 bp PCR fragment obtained on the DNA of strain 204 was used in further work.The fragment was cloned into the pT-Adv vector (Clontech, USA) according to the manufacturer's instructions and its nucleotide sequence was determined by automatic sequencing on an ABI 373 DNA instrument Sequencer using P set erkin-Elmer "fluorescent dye Cycle sequencing kit." Comparison of the obtained nucleotide sequence with the DNA sequence of the LANS gene of Erw. chrysanthemi (GenBank Acc # A 14577, ECRY-LANS) showed that the level of homology between the selected fragment and the corresponding central fragment of the ECRY-LANS gene is 85% (figure 4). Thus, the resulting fragment with a high probability corresponds to the Central part of the desired ECAR-LANS gene.

Example 2. Determination of the coding sequence of the ECAR-LANS gene
To isolate and determine the primary structure of flanking regions of the ECAR-LANS gene that overlap the coding amino acid sequence of this protein, the “inverted PCR” strategy was used. Based on the sequencing data of the central part of the ECAR-LANS gene of strain 204, two primers — 204F and 204R — were designed, the structure and position of which are shown in Table 2 and in FIG. 5.

 Genomic DNA preparations of strain 204 in an amount of 2-4 μg were hydrolyzed with various restriction endonucleases - BamHI, BglII, Bspl201, EcoRI, NaeIII, HindIII, KpnI, PstI, SalI, Sau3A, SpeI, Xba, XhoI individually or in combination of two enzymes that form after cleavage of DNA with identical "sticky ends", for example BamHI + BglII, SalI + XhoI, XbaI + SpeI. After incubation for 10-16 hours at the desired temperature, the restriction endonucleases present in the mixture were inactivated by heating (10-15 min at 65 degrees), 1/10 of the solution containing 100 mM DTT and 10 mM ATP, 1-2 units of T4 DNA was added ligases and incubated for another 10-12 hours at +4 degrees. The obtained samples were deproteinized by treatment with a mixture of phenol with chloroform (1: 1) and precipitated with alcohol according to standard methods / 14 /. DNA was dissolved in a volume of 50 μl and 10-15 μl was used in a PCR reaction with 204F / 204R primers, which was performed using Taq polymerase or the Expand Long Template PCR system kit (Roche Diagnostics GmBh, Mannheim, Germany). PCR conditions and characteristics of the obtained products are shown in table 2.

 Samples obtained on Sau3A, HaeIII, EcoRI, SalI matrices were isolated from agarose gel using the Wizard PCR prep purification system kit manufactured by Promega (USA) in accordance with the manufacturer's instructions and their nucleotide sequence was determined by automatic sequencing on an ABI 373 instrument. DNA Sequencer. Synthetic oligonuleotides 204F and 204R were used as primers. The complete sequence was restored by constructing a "contig" using the program Oligo / 15 /. Comparison of the obtained sequence with the ECRY-LANS gene structure showed that the level of homology between these sequences is 85% (Fig.6). Figure 1 shows the nucleotide sequence and deduced from it the complete amino acid sequence of the ECAR-LANS gene. A comparison of the amino acid sequences of ECAR-LANS and ECRY-LANS (Fig. 7) confirmed a high degree of homology between the two proteins. The degree of identity was 72%, the degree of similarity was 87%.

 Example 3. Isolation and cloning of a fragment of the nucleotide sequence of the ECAR-LANS gene encoding the complete amino acid sequence of this protein.

 The genomic DNA preparation of strain 204 was used in PCR reaction with oligonucleotides N204 and C204, the structure and specificity of which are shown in Table 1. The primers used limit the ECAR-LANS gene fragment including the ATG translation initiation codon located at position + 1 of the sequence shown in FIG. 6, and the TAA termination codon located at position + 1045 of the same sequence. Thus, a full-sized specific amplification reaction product should include the complete coding sequence of the ECAR-LANS gene. The following amplification conditions were used: initial denaturation - 94, 2 min, then 30 cycles of amplification - 94, 40 s, 50, 40 s, 72.1 min 30 s. The reaction mixture was separated by electrophoresis on a 1% agarose gel and the reaction product — a single fragment about 1100 bp in size — was detected. The fragment was isolated from the gel using the Wizard PCR preps Purification System kit (Promega, USA) and cloned into the pT-AdV vector (Clontech, USA), as described in Example 1. Plasmid DNA was isolated from the obtained individual transformant clones by standard methods and analyzed it by hydrolysis with restriction enzyme EcoRI and separation of reaction products in 1% agarose gel. Since the EcoRI restriction enzyme sites flank on both sides the cloning site of the PCR fragment in the pT-AdV vector, the appearance of a fragment of about 1 TPN in the EcoRI hydrolysates of the studied plasmid DNA indicates the presence of the desired insert in recombinant clones. A total of 12 clones that were "positive" in this test were selected and used in further work. To prove that the obtained clones contain a fragment of the ECAR-LANS gene, plasmid DNA was used in “PCR test reactions” using combinations of primers listed in table 3. The presence of PCR products with the characteristic sizes shown in table 3 confirmed that the resulting clones contain the desired fragment.

 Example 4. Construction of a recombinant plasmid pBADLANS for the expression of ECAR-LANS in Escherichia coli cells.

To prove that cloned in paragraph 3. DNA fragments are capable of directing the synthesis of functionally active ECAR-LANS in E. coli cells; plasmids containing the ECAR-LANS gene fragment under the control of the PBAD promoter vector pBAD24 were constructed. To obtain an “insert”, 2 μg of DNA from each of the 12 “positive” clones selected according to the procedure described in Section 3. were combined in one tube and the total DNA was hydrolyzed with EcoRI restrictase enzyme. A “total” EcoRI fragment of about 1 kb corresponding to the ECAR-LANS gene fragment was isolated from the agarose gel. To obtain a “vector”, 2 μg of plasmid DNA pBAD24 was linearized at the EcoRI site, treated with calf intestine phosphatase to prevent the self-loopback of the vector, and a 4.5 kb fragment was isolated from the agarose gel. 20 ng of the “vector” was ligated with 50 ng of the “fragment” using T4 DNA ligase under standard conditions and the competent cells of E. coli XL1 strain were transformed with the mixture. Individual transformant clones were analyzed for their ability to direct the synthesis of functionally active ECAR-LANS. For this, individual colonies (30 pieces) were grown on LB medium containing 100 μg / ml ampicillin up to OD600 ~ 1.0 in a volume of 5 ml. After that, L-arabinose was added to the cultures to a final concentration of 0.2% to induce the pBAD promoter and incubation was continued for 2 hours. Aliquots of the cell suspension of the grown individual transformant cultures were used to determine L-asparaginase activity using a micromethod developed for this purpose. The method is based on the periplasmic localization of ECAR-LANS expressed in E. coli cells, due to which the substrate (asparagine) freely penetrates the cell and interacts with the enzyme. Cell suspensions (50 μl) were placed in a 96-well plate and incubated (37 ^° C; 20 min) with 50 μl of L-asparagine (30 mg / ml in 0.0125 M borate buffer, pH 9.5). At the end of the incubation, 50 μl of Nessler's reagent was added to the wells. Asparaginase activity is assessed visually by the intensity of developing staining. Control samples contain either bacterial cells or a solution of L-asparagine. Thus, of the 30 analyzed clones, 5 "positive" were identified. Selected positive clones were further analyzed for the presence of an insert with an ECAR-LANS gene fragment. To do this, plasmid DNA was isolated, hydrolyzed with EcoRI restriction enzyme and the presence of hydrolysis products of 4.5 TPN ("vector") and 1.1 TPN ("fragment") was detected. One of the selected clones was introduced by transformation into E. coli strain BL21 (DE3). To quantify the level of ECAR-LANS expression in the resulting recombinant strains, individual clones of BL21 (DE3) / pBADLANS transformants were grown on LB medium in a volume of 50 ml in 500 ml flasks at a temperature of 37 ^° C A ₆₀₀ = 0.6-0.7 OE . Then, the inductor L-arabinose was added to a concentration of 0.2% and incubated for another 22 hours, taking periodically samples of the culture fluid to determine the growth of the culture and enzyme activity in bacterial cells / 16 /. The data obtained are summarized in table 4. As can be seen from the data presented, the specific activity of the enzyme reaches a maximum of about 55 IU / mg protein after 4 hours of incubation, which is about 50-60 times higher than that of the original wild strain Erw.carotovora 204. Thus , in these experiments, a fairly high level of expression of recombinant Erw.carotovora asparaginase (8-10%) of the total cell protein was achieved. A comparison of the obtained data with the previously known literature on the characteristics of recombinant strains of E. coli producing bacterial asparaginases suggests that the production level of the recombinant ECAR-LANS in the strain we created is about 1.5 times higher than the productivity of the best of the previously developed samples / 8 / . The physical and genetic map of the obtained plasmid is shown in figure 2.

Claims (2)

1. The gene of L-asparaginase Erwinia carotovora with the coding sequence of SEQ ID No. 1. 2. The strain Escherichia coli VKPM No. B-8174 containing the plasmid DNA pBADLANS, the producer of L-asparaginase Erwinia carotovora.

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