RU2071501C1 - Vector pel5a designated for foreign dna expression - Google Patents

Abstract

FIELD: biotechnology, genetic engineering. SUBSTANCE: vector $$$ has operon consisting of gene encoding recombinant protein and gene encoding lysozyme. Simultaneously with synthesis of recombinant protein lysozyme is synthesized that decomposes polysaccharide envelope of E. coli cells. Vector can be used for plasmid and the corresponding strain-producers of recombinant proteins construction and for proteins purification. EFFECT: simplified purification of water-insoluble recombinant protein agglomerate. 1 dwg

Description

 The invention relates to the field of biotechnology, genetic engineering and can be used to obtain plasmids synthesizing recombinant proteins.

Large amounts of recombinant proteins can be synthesized in Escherichia coli cells carrying recombinant gene expression systems based on phage lambda promoters (see, for example, H. Bernard and DR Helinski. Methods Enzymol. Vol. 68, pp. 482-492, 1979). Transcription in such systems is regulated by the binding of the temperature-sensitive repressor of the lambda phage encoded by the mutant gene to Its857 with the operator site. When cells grow at a temperature of 30-32 ^o C, the repressor binds to the operator and blocks expression. When the growth temperature is increased to 42 ^° C, the repressor is inactivated, which allows the RNA polymerase to contact the promoter and begin transcription of the gene. Significant amounts of protein can be synthesized as a result of intense transcription and subsequent translation.

Recombinant plasmid DNA pEX2 and pEX3 are known for expressing recombinant proteins in all three reading frames (KK Stanley and JP Luzio. The EMBO Journal, vol. 3, pp. 1429-1434, 1984). DNA sequences for expression can be inserted into the polylinker located at the 3'-end of the lacZ gene. In these plasmids P _{R, the} lambda bacteriophage promoter provides expression of DNA sequences, and the product makes up a significant portion of the total bacterial protein. The expressed protein in the cell accumulates in the form of water-insoluble agglomerates.

 However, these vectors have a drawback: in order to isolate water-insoluble agglomerates of a recombinant protein, it is necessary to destroy the bacterial cell wall.

 The pEX1 vector is selected as a prototype for the preparation of the pEL5a vector. The advantage of the claimed vector pEL5a is that as a result of using an operon consisting of a gene encoding a recombinant protein and a lysozyme gene (Fig. 1), simultaneously with the synthesis of a recombinant protein, lysozyme is synthesized that destroys the polysaccharide membrane of E. coli, which greatly simplifies purification of water-insoluble agglomerates of a recombinant protein.

The essence of the invention lies in the fact that a vector pEL5a of 6.4 thousand base pairs (kbp) is constructed, consisting of the following elements (Fig. 1):
-Xbal-Xbal-fragment of plasmid DNA of the bacterial vector pEX1 size of 5.8 bp which contains a fusion cro-lacZ, a gene encoding a fusion protein under the control of the promoter P _{R of the} bacteriophage lambda, a polylinker at the 3'-end of the lacZ gene;
-BamH1-BamH1 DNA fragment of the plasmid pLysS (ACY Chang and SN Cohen. J. Bacteriol. Vol. 134, pp. 1114, 1978), containing the bacteriophage T4 lysozyme gene and having a size of 0.6 kb.

To construct plasmid pEL5a, the DNA of plasmid pEX1 was hydrolyzed with restriction enzyme Xbal and two nucleotides were added at the sticky ends using PollK DNA polymerase. At the same time, a BamH1 restriction of 0.6 size is obtained. about. from plasmid pLysS (ACY Chang and SN Cohen. J. Bacteriol. vol. 134, pp. 1141, 1978), containing the bacteriophage T4 lysozyme gene and having two PollK-linked nucleotides in sticky ends. The pEX1 fragment is ligated with a DNA fragment containing the bacteriophage T4 lysozyme gene. The E. coli cells containing the temperature-sensitive lambda bacteriophage gene with Its857 in the chromosome are transformed using a ligase mixture, for example, cells of strain PLT90. Transformants are plated on medium with ampicillin at 30 ^° C. Clones containing recombinant plasmids with the lysozyme gene in the desired orientation are selected by lysis assay after induction of protein synthesis. The expression of recombinant proteins is induced by increasing the temperature to 42 ^o C. In the clones producing lysozyme, after the induction of protein synthesis and the subsequent addition of chloroform, cell lysis occurs. From the selected clones, plasmid pEL5a is isolated by standard methods. Vector DNA is stable when stored in 10 mM Tris, pH 8.0, 1 mM EDTA at -20 ^o C.

 The invention is illustrated by the following examples.

 Example 1. Obtaining a vector plasmid pEL5a with an operon expression and lysis system.

Bacterial cells of E. coli PLT90 containing plasmid pEX1 (KK Stanley and JP Luzio. The EMBO Journal, vol. 3, pp. 1429-1434, 1984) were grown in 50 ml of 2YT broth (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl per 1 liter of water) containing 100 μg / ml ampicillin to a titer of 10 ₉ cells / ml.

Cells are pelleted by centrifugation, resuspended in 2 ml of solution (50 mM glucose, 25 mM Tris-HCl, pH 8.0, 10 mM EDTA, 5 mg / ml lysozyme) and incubated for 5 min at room temperature. Then add 4 ml of a solution of 0.2 M NaOH, 1% sodium dodecyl sulfate, mix, incubate for 10 min in ice, add 3 ml of chilled 5 M potassium acetate solution, pH 4.8, mix, leave for 10 min in ice, the precipitate formed separated by centrifugation. 0.6 volumes of isopropyl alcohol are added to the supernatant and incubated for 15 minutes at room temperature. The precipitate was collected by centrifugation, resuspended in 0.5 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and an equal volume of saturated sodium acetate solution was added. After incubation for 30 minutes at minus 20 ^{° C., the} precipitate is removed by centrifugation, and 0.6 volumes of isopropanol are added to the supernatant and left for 1 hour at room temperature. The precipitate was collected by centrifugation, washed with 70% ethanol and resuspended in 100 μl of TE8 buffer.

Plasmid DNA (1 μg pEX1) is treated with Xba1 restriction endonuclease (10 units) in buffer A (50 mM Tris-HCl, pH 7.6, 10 mM MgCl ₂ , 1 mM dithiothreitol, 100 mM NaCl, 4 mM spermidine) for 2 hours. Analysis of the completeness of hydrolysis is carried out using electrophoresis. Using the Klenov fragment of E. coli DNA polymerase, two of the four nucleotides in the sticky Xba1 ends are completed. Proteins are removed by phenolic extraction, DNA is precipitated with ethanol and resuspended in 5 μl TE8.

To obtain a DNA fragment with the lysozyme gene, 10 μg of pLysS plasmid DNA is restriction (ACY Chang and SN Cohen. J. Bacteriol, vol. 134, pp. 1141, 1978) with restriction enzyme BamH1. Using a Klenov fragment of E. coli DNA polymerase, two of the four nucleotides are added at the sticky BamH1 ends. Fragment -BamH1-BamH1- with partially completed sticky ends measuring 0.6 bp isolated by electrophoresis by sorption on DE81 paper. The paper is washed, incubated for 30 min at 80 ^o C in a buffer of 2 M sodium acetate, 50 mm Tris-HCL, pH 7.5, 10 mm EDTA. DNA transferred to the solution is removed from the paper by centrifugation, 2 volumes of ethanol are added to the solution. After incubation at -20 ^° C for 1 hour, the precipitate was collected by centrifugation and dissolved in 5 μl of TE8 buffer.

0.5 μg of the pEX1 vector DNA fragment is mixed with 0.2 μg of DNA containing the bacteriophage T4 lysozyme gene. The connection of the fragments is carried out using 10 units Phage T4 DNA ligases in buffer: 30 mM Tris-HCl, pH 7.2, 10 mM magnesium chloride, 2 mg / ml gelatin, 1 mM spermidine, 0.1% mercaptoethanol, 0.2 mM ATP at 20 ^° C within 2 hours.

The resulting mixture was transformed with E. coli PLT90 cells containing clts857 repressor. For this, the overnight cell culture was diluted with LB medium with 10 mM MgSO ₄ in a ratio of 1: 100 and grown with aeration to a density of A550 = 0.3 OE. After 15 minutes of cooling at 0 ^{° C., the} cells were collected by centrifugation, suspended in 1/3 of the original volume with 30 mM potassium acetate buffer, pH 5.8, 100 mM RbCL, 50 mM MnCl ₂ , 15% glycerol, and left for 30-60 minutes on ice. Cells are harvested by centrifugation, resuspended in 1 / 12.5 of the original volume in 10 mM MOPS buffer, pH 6.8, containing 10 mM RbCl, 75 mM CaCl ₂ , 15% glycerol and incubated for 15 min on ice. Ligated DNA fragments are added to the cell suspension, incubated for 40 min at 0 ^° C, then 2 min at 34 ^° C, 2-3 min at 0 ^° C, diluted 5 times with 2YT medium, grown 30 min at 30 ^° C and seeded 2YT agar medium with ampicillin (50 mg / ml).

Clones containing recombinant plasmids with the lysozyme gene in the desired orientation are selected by lysis assay after induction of protein synthesis. The expression of recombinant proteins is induced by increasing the temperature to 42 ^o C. In the clones producing lysozyme, after the induction of protein synthesis and the subsequent addition of chloroform, cell lysis occurs. The plasmid pEL5a is isolated from the selected clones by standard methods. Vector DNA is stable when stored in 10mM Tris, pH 8.0, 1 EDTA at -20 ^o C.

 Example 2. The use of the vector pEL5a to obtain recombinant proteins in the form of water-insoluble agglomerates.

PLT90 cells containing the plasmid pEL5a were inoculated in 1 ml of LB medium with 100 μg / ml ampicillin and grown overnight at 30 ^o C. The night culture was diluted in 2 ml of the same medium in a ratio of 1: 100 and grown with aeration to 0.2 OE at 550 nm, then raise the temperature to 42 ^o With for the induction of synthesis of fusion protein and grow for another 2 hours. Then add chloroform to 1% and incubate at 37 ^o With another hour. The lysate is then centrifuged, the precipitate is resuspended in 75 μl of TE8 buffer, and an equal volume of electrophoresis buffer (160 mM Tris-HCl, pH 6.8, 20% glycerol, 4% sodium dodecyl sulfate, 4 mM EDTA, 6% mercaptoethanol, 0, 05% bromophenol blue). Cellular proteins are analyzed by Lammley polyacrylamide gel electrophoresis (UK Lammli. Nature, vol. 227, pp. 680-685, 1970). After electrophoresis, the proteins in the gel are fixed and Coomassie stained with bright blue R-250.

 An analysis of the results shows that as a result of the action of lysozyme, the bacterial membrane is destroyed and water-soluble cellular proteins are released into the medium, which is manifested in the weakening of minor protein bands. In the control, in the case of using the plasmid pEX1, there is no such effect.

 Thus, the invention allows to obtain water-insoluble agglomerates of recombinant proteins without the use of additional methods of destruction of cell walls.

Claims (1)

The vector pEL5a, designed for the expression of foreign DNA, size 6.4 kb, containing: XbaI-XbaI fragment of the plasmid DNA of the bacterial vector pEXI size 5.8 kb with a fused cro-LacZ gene encoding a protein under the control of the promoter P _{R of the} bacteriophage lambda and polylinker at the 3'-end of the LacZ gene; BamHI-BamHI DNA fragment of plasmid pLys S with the bacteriophage T4 lysozyme gene of 0.6 kbp unique restriction sites for DNA cloning at the 3'-end of the LacZ, SmaI, BamHI, SalI, PstI gene; operator O _R and promoter P _{R of} bacteriophage λ phage transcription terminators fd; ampicillin resistance genetic marker; host spectrum - Escherichia coli bacteria with clts 857 gene of bacteriophage lambda.