Recombinant phage m13 dna pol t7 and recombinant strain of phage m13 - producer rna polymerase of phage t7

 

(57) Abstract:

Usage: biotechnology, genetic and protein engineering. The inventive by introducing a DNA vector phage MMR unique BamH1 restriction site Sau3A-Sau 3A fragment of plasmid PAR 1219, containing the gene for RNA polymerase of phage T7, constructed recombinant DNA M13polT7, on the basis of which the resulting strain of phage M13 PMBC PH 714 for synthesis of DNA polymerase T7 exit 2106units act/g wet biomass. 2 S. p. f-crystals., 2 Il.

The invention relates to biotechnology, in particular genetic engineering, and is a recombinant phage DNA M13polT7, containing the gene for RNA polymerase of phage T7 and strain of phage M13polT7 producing RNA polymerase of phage T7.

RNA polymerase of phage T7 is widely used to obtain RNA probes for blot-hybridization; obtaining biologically active RNA, studying the processing of eukaryotic RNA exon-intron mapping of genomic DNA [1] in Addition, the enzyme can be used for labeling RNA with radioactive or biotinylated of nucleosidase, and in certain systems for sequencing nucleic acids [2]

However, the strain of E. coliHMS174 transformed this loft is">

Recently as cloning vectors have been actively using filamentous phages of E. coli. Created various vector derivatives of phage: M13, fd and fi [4] the Advantage of vectors of this type is that filamentous phage during infection is not are lysed bacterial cells, and chopinot replicative form ragovoy DNA in the cell reaches 200-300 molecules that delivers a high dose of the cloned gene, and hence a sufficiently high yield RNA polymerase of phage T7. In addition, phage DNA is easier to allocate and use compared to plasmid DNA.

Known phage M13polT7-1, used in a vector system capable of expression of foreign genes in E. coli cells under the control of a late promoter of phage T7.

This vector system is based on the separate introduction of genes into bacteria by pre-transformation of E. coli cells with plasmid with the expressed gene with subsequent infection in their bacteriophage M13polT7-1, which acts only as a carrier of the gene for RNA polymerase, but is not its producer.

Known recombinant plasmid PAR1219, containing the gene for RNA polymerase of phage T7.

The technical task proposed is the construction of recombinant phage M13polT7, providing an increased synthesis of the enzyme RNA polymerase of phage T7 in bacterial cells.

Phage DNA M13polT7 containing the DNA fragment encoding the RNA polymerase of phage T7 has a molecular weight of 6.6 MDA (size 9949 N. p.) and consists of the following elements:

vector BamHI fragment of phage M13mp10, size 7244 N. p.

Sau3A-Sau3A fragment of plasmid PAR1219, containing the gene for RNA polymerase of phage T7, size 2705 N. p.

plac-lacuv5 promoter;

is:

a unique restriction site: BamHI;

marker gene: lacZ.

The strain of phage M13polT7 has the following cultural and morphological properties.

Morphology of negative colonies on solid medium: forms clear plaques with a diameter of 2 mm.

Functional properties: nelisiwe phage.

The optimum growth: (37+1)oC on YT environment.

The strain deposited at the all-Union collection of industrial microorganisms Institute "Vniigenetika" number VKPM PH 714.

The essence of the invention lies in the fact that the DNA fragment encoding the RNA polymerase of phage T7, injected into the phage vector DNA of filamentous phage M13. The obtained recombinant phage provides a synthesis of the RNA polymerase of phage T7 in cells of E. cI injected fragment from plasmid PAR1219 [3] encoding RNA polymerase of phage T7.

The resulting phage M13polT7 in E. coli cells provides a synthesis of the RNA polymerase of phage T7 (25-30% of total protein).

Thus, for the first time obtained phage DNA, which infected her cells E. coli fusion protein with the properties of RNA polymerase of phage T7, due to gene expression RNA polymerase of phage T7.

In Fig.1 shows the schematic construction of phage M13polT7 with the following symbols: polT7 gene RNA polymerase of phage T7, SD binding site of the ribosome, plac-lacuv5 promoter, lacZ part galactosidase gene; Fig.2-densitogram electrophoretic separated lysates of E. coli cells infected with phage M13polT7. The shaded peak corresponding protein polT7.

The essence of the invention disclosed in the following examples.

Example 1. Method of constructing recombinant phage M13polT7. 1 μg DNA vector phage M13mp10 [6] decompose the restriction enzyme BamHI (10 units) in a buffer containing 10mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MgCl2,1mM dithiothreitol. The reaction is carried out for 1 h at 37oC. analysis of the completeness of the hydrolysis is carried out using electrophoresis. The drug deproteinized using phenol extraction, the DNA precipitated with ethanol and resuspended in TE-buffer containing 10mM Tris-HCl pH 8.0 and 1 mM EDTA is up with Sau3A hydrolysis of plasmid pAR1219 [3] the Selection of individual fragment is carried out by electrophoretic separation in a 6% polyacrylamide gel followed by Electrosila.

Ligation mixture hydrolyzed phage M13mp10 (0.1 µg) and fragment encoding gene RNA polymerase (0.1 ág),performed using DNA ligase of phage T4 (4 units) in a volume of 30 ál (incubation are within 10 h at 12oC).

The resulting phages that do not have a blue color on indicator plates from Ygal (0.1 ág/ml) and IPTG (1 Mm) analyze restriction fragments length polymorphism analysis of replicative forms of these phages using the restriction enzyme HaeIII.

Recombinant phage DNA, which has in its composition the fragment containing the gene for T7 RNA polymerase in the correct orientation under the control of the lacuv5 promoter), marked M13polT7.

Example 2. Gene expression RNA polymerase of phage T7 in the composition of the phage M13. To study the gene expression RNA polymerase of phage T7 E. coli cells D5F infecting phages: M13polT7 and M13mp10. E. coli cells D5F pokasivaut to optical density (OD) of 0.6-0.7% (590 nm) in LB medium at 37oC. Then contribute phage M13polT7, and in the control experiment, the phage M13mp10; and an inductor - IPTG (isopropyl-D thiogalactoside) (0.1 mm). The ratio of the cell: phage - 1:1 (multiplicity of infection).

Suspension cultured for 6 h, which allows to obtain the maximum yield of the target protein in the case of phage M13polT7.

On electrophoregram separation browarnik vector phage M13mp10.

Determination of enzyme activities carried out, using as specific template DNA of phage T7.

Based on the data of electrophoresis level of product RNA polymerase synthesized by recombinant phage, approximately 25-30% of the total protein.

Example 3. The selection and titration of the phage. To the culture fluid after separation of infected cells add 0.25 to the original volume of PEG solution (containing polyethylene glycol 6000 20 g, NaCl 10 g, water to 100 ml) and left for 15 min at 20oC. the Phage was separated by centrifugation (15 min) and resuspended in a buffer solution containing 20 mm Tris-HCl pH 7.5, 20 mm NaCl, 1mm, ADEA (1/10 of the original volume). Titration of phage performed by standard methods. Usually get the drugs phage with a title 10-10 PFU/ml.

Example 4. Purification of recombinant RNA polymerase of phage T7. E. coli cells DH5F infected with the phage M13polT7, precipitated by centrifugation. 6 g of biomass suspended in 160 ml of 50 mm Tris-HCl buffer pH of 7.9,containing 2 mm EDTA, 0.1 mm of dithiothreitol, 200 mm NaCl, 5% glycerol and 10 mg of lysozyme. Cells destroy ultrasound for 1 min and centrifuged for 30 min at 1000 rpm To the supernatant add solution streptomycinresistant to a final concentration of 0.2% at 0 rpm for 20 min.

The supernatant containing the RNA polymerase of phage T7, add ammonium sulfate to 50% saturation. The suspension is centrifuged for 30 min at 10000 rpm dissolved in 40 ml of 10 mm potassium phosphate buffer pH 8.0, containing 10 mM-mercaptoethanol, 1 mM EDTA, 12% glycerine (buffer A) and 0.15 M NACl, and subjected to dialysis against the same buffer for 12 h

The desalted enzyme solution is applied on a column (1.51.5) phosphocellulose P11.

RNA polymerase of phage T7 elute column with a gradient of NaCl (0.25 M To 0.5 M) in the buffer And RNA polymerase eluted at NaCl concentration of 0.25 M To 0.4 M. the Fractions containing the enzyme are combined and applied to a column (1,5x6 cm) with blue dextran-separate. The column is washed once with 40 ml of buffer a containing 0.25 M NaCl, then with 20 ml of buffer a containing 1 mm GTP and 1 mm ATP.

The enzyme elute 0.55 M potassium phosphate buffer pH 8.0, containing 1.5 mm dithiothreitol, 40 mm EDTA and 12% glycerol.

The fractions containing RNA polymerase of phage T7, collect and cialiswhat against buffer A, and then concentrate against 10 mm potassium phosphate buffer pH 8.0, 10mM b-mercaptoethanol, 50% glycerol and 180 mm KCl.

Thus, in the scheme of purification receive 12106units of the act. homogeneous RNA in the S="ptx2">

With 1 g of wet biomass receive 2106units of the act. homogeneous enzyme.

References:

1. KKrieg,P. A. And Melton,D. A. Methods Enzymoll.155,397(1987).

2. Axelrood,V. D. Rramer,F. R. Biochemistry 24,5716(1985).

3. Davanloo,P. et al.Proc.Natl.Acad.Sci:USA 81,2035(1984).

4. Messing, J. and Vieira, J., Gene,1982,v.19.p7269-276.

5. A. A. Il'ichev, N. In.Melamed, A. I. Zakabunin and other DAN, 1988,I. 303,N2,c. 496-498.

6. Single-atranded DNA Phages (1978). eds.Dehardt D. T. Dressler D. H. and Ray, D. S. Cold Spring Harbor Laboratory,ID 11724 Cold Spring Harbor,New York.

1. Recombinant phage M13 DNA polT7 sized 9949 p. O. molecular weight of 6.6 Hmm, consisting of a Bam HI DNA fragment phage vector M13 mp 10 size 7244 p. O. Sau Sau 3A 3A DNA fragment size 2705 p. O. from plasmid pAR 1219 encoding RNA polymerase of phage T7; plac lacuv 5 promoter,

and containing marker gene lac Z; unique restriction site Bam HI.

2. Recombinant strain of phage M13 PMBC PH 714 producer RNA polymerase of phage T7.

 

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