RU2653750C1 - Method for obtaining heterotetrameric recombinant streptavidin from periplasm of escherichia coli - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, gene engineering and biotechnology, in particular to a method for producing heterotetrameric recombinant streptavidin. Present method involves the creation of two plasmids, one of which encodes streptavidin of the mutant type and the other is wild-type streptavidin, transformation of the E. coli cells by said plasmids, induction of streptavidin synthesis, isolation of the periplasmic cell fraction and purification of heterotetrameric streptavidin on an affinity sorbent. Method is characterized in that in one embodiment, wild-type streptavidin is linked to an affinity tag, and mutant-type streptavidin is coupled to PelB leader peptide, in the second case, mutant-type streptavidin is linked to an affinity tag, and wild-type streptavidin is coupled to PelB-leader peptide.

EFFECT: present invention makes it possible to obtain heterotetrameric recombinant streptavidin with high efficacy.

10 cl, 12 dwg, 9 ex

Description

The invention relates to the microbiological industry and genetic engineering, and is a method for producing heterotetrameric streptavidin from the periplasmic space of a cell. The proposed method includes the creation of two plasmids obtained on the basis of the vectors pET22b + and pET28a + and encoding the monomers of mutant streptavidin (unable to bind biotin) and wild-type streptavidin, respectively, and the creation of a producer strain E. Coli, providing gene expression in the cell. Obtaining the periplasmic fraction of the cells of the bacterial strain and protein purification on nickel agarose. Heterotetrameric streptavidin is used in various methods of diagnosing diseases, for example, in methods such as enzyme-linked immunosorbent assay and immunohistochemistry.

Currently, the main source of streptavidin is the producer S. avidinii ATCC 27419, which has a high natural resistance to antibiotics and requires a long cultivation time. In addition, when streptavidin is isolated from S. avidinii, the target protein is often contaminated with biotin [Proc. Natl. Acad. Sci. 1980. V. 77. P. 4666-4668; Biochem. Biophys. Methods 1986. V. 13. P. 103-112; J. Immunol. Methods 1988. V. 113. P. 83-91].

PCT / US 2006/016480, published August 13, 2009, proposes a method for expressing streptavidin into a culture medium, which complicates the process of protein isolation due to the large volumes of culture medium driven through a nickel column.

A known method of obtaining the construction of an expression plasmid for the production of streptavidin in the periplasmic space (pSAV27) [RU 2153535, publ. 07.27.2000]. The preparation of a plasmid includes: isolation from S. avidinii M170791 of a nucleotide sequence encoding a streptavidin leader peptide and a mature protein (prostreptavidin) with the introduction of restriction sites; construction of a high copy pSAV27 plasmid with an ampicillin marker; transformation of E. coli strain JM110 with the resulting construct; growing producer strain on LB medium; lysing the resulting biomass to isolate the periplasmic fraction; determination of the biological activity of streptavidin by separation in the PAGE of the products of the binding of streptavidin with a synthetic biotinylated oligonucleotide. Compared with the above method of isolating protein from inclusion bodies, this method has a significant advantage. Plasmid pSAV27, carrying the leader peptide sequence together with the nucleotide sequence of the homotetrameric streptavidin, selectively expresses the protein in the periplasmic space, where it is localized in a biologically active state. The disadvantage of the method for producing streptavidin described in RU 2153535 is that the homotetrameric streptavidin obtained in this work is capable of binding four biotin molecules, which subsequently leads to conglomeration and loss of physiological properties of the transported molecule.

The closest set of essential features to the proposed method is a method for heterotetrameric streptavidin, described in US patent US 8586708 B2. The authors propose obtaining monovalent streptavidin in several stages: at the first stage, two plasmids are created that encode the monomers of mutant streptavidin and wild-type streptavidin, then different E. coli cells are transformed with these constructs and the transformates are separately cultured in nutrient medium at 37 ° C. Protein synthesis is induced by the addition of isopropyl-β-D-1-thiogalactopyranoside. At the second stage, the mutant (incapable of binding biotin) and wild-type streptavidin (capable of binding biotin) are separated separately from the inclusion bodies of E. Coli cells. At the third stage, streptavidins are denatured in 6M Gu-HCl to obtain both types of streptavidin monomers, a mixture of wild type mutant and streptavidin is proposed to be mixed in a ratio of 3: 1, respectively, and at the final stage, the resulting protein is renatured in PBS (phosphate buffer solution). The resulting protein is concentrated by precipitation using ammonium sulfate and then subjected to double dialysis against the FBI to purify wild-type streptavidin from biotin. Then heterotetrameric streptavidin is isolated on a nickel-agarose column. The method for producing a heterotetrameric protein described in US patent US 8586708 B2 has several disadvantages: this process is time-consuming and resource-intensive, the yield of the target protein is reduced and the biological properties of the protein are irreversibly lost. This leads to an increase in the cost of streptavidin. The reason for these shortcomings is the presence of additional stages of isolation, denaturation and renaturation of protein homotetramers to obtain heterotetrameric streptavidin.

The technical problem solved by the invention is the creation of such a method for producing heterotetrameric recombinant streptavidin from the periplasm of E. coli, which would reduce the cost of the target protein without losing the biological properties of the molecules.

The technical result is to increase the efficiency of the process of obtaining heterotetrameric streptavidin, consisting simultaneously of monomers of mutant streptavidin and wild-type streptavidin, by eliminating additional stages of protein isolation and denaturation-renaturation.

It is proposed to create two genetic constructs, one of which encodes a mutant streptavidin monomer, and the other a wild-type streptavidin monomer. These constructs transform the same E. coli cells, which makes it possible to obtain the target heterotetramer directly in the cell, bypassing additional stages of protein isolation and denaturation-renaturation in vitro.

Thus, the technical result is achieved due to the fact that in the method for producing heterotetrameric recombinant streptavidin from E. coli periplasm, including the creation of two plasmids for the expression of streptavidins, transformation of the obtained E. coli cell plasmids, culturing cells in a culture medium, inducing isopropyl synthesis of streptavidins β-D-thiogalactopyranoside, isolation of streptavidin from the periplasmic space and purification of heterotetrameric streptavidin on an affinity sorbent,

offered

- as the first plasmid, based on the initial vector, create a plasmid that expresses wild-type streptavidin linked to an affinity tag and has an antibiotic resistance gene of the aminoglycoside group,

- as a second plasmid, based on the initial vector, create a plasmid that expresses mutant streptavidin linked to the pelB leader

a peptide and incapable of binding biotin having a penicillin antibiotic resistance gene,

- carry out the transformation of the same E. coli cells with the two indicated plasmids, followed by culturing these transformed cells in a culture medium containing both an antibiotic of the penicillin series and an antibiotic of the aminoglycoside group,

or proposed

- as the first plasmid based on the original vector to create a plasmid that provides the expression of mutant streptavidin linked to an affinity tag and is not capable of binding biotin, having an antibiotic resistance gene of the aminoglycoside group,

- as a second plasmid, based on the initial vector, create a plasmid that expresses wild-type streptavidin linked to a pelB leader peptide having the penicillin antibiotic resistance gene,

- carry out the transformation of the same E. coli cells with the two indicated plasmids, followed by the cultivation of these transformed cells in a culture medium containing both an antibiotic of the penicillin series and an antibiotic of the aminoglycoside group.

Additional differences of the proposed method are:

- Nickel agarose is preferably used as an affinity sorbent;

- it is preferable to use ampicillin as an antibiotic of the aminoglycoside group;

- as an antibiotic of the penicillin series, it is preferable to use kanamycin;

- the vector pET28a + can be used as a vector for the first plasmid;

- the vector pET22b + can be used as a vector for the second plasmid;

- as a strain for transformation of E. coli cells, E. coli strain BL21 (DE3) can be used;

- mutant streptavidin not capable of binding biotin, or wild-type streptavidin linked to a fusion peptide;

- the TAT peptide is used as a fusion peptide;

- as an affinity tag, it is preferable to use the oligohistidine sequence.

The invention is illustrated by the following figures.

In FIG. 1 is a schematic representation of the synthesis of monovalent streptavidin with a TAT peptide in an E. coli strain. In FIG. Figure 2 shows a schematic representation of the synthesis of bivalent streptavidin with a TAT peptide in E. coli strain. In FIG. Figure 3 shows a schematic representation of the synthesis of tetravalent streptavidin with a TAT peptide in E. coli strain. In FIG. Figure 4 shows a schematic representation of the synthesis of the wild-type streptavidin homotetramer with a TAT peptide in E. coli strain. In FIG. Figure 5 shows a schematic representation of the synthesis of homotetramers of mutant streptavidin in the E. coli strain. In FIG. Figure 6 schematically shows how restriction endonucleases NcoI and XhoI cut the pET22b (+) vector to form two “sticky ends”. In FIG. 7 schematically shows a process for producing SAD products using f-SA-NCO and r-SA-stop-XHO oligonucleotides. The nucleotide sequence of SAD is indicated in black on the scheme. On the right in the diagram is indicated the final amplification product - SAD. In FIG. Figure 8 schematically shows the ligation process of the pEP22b (+) / NcoI / XhoI construct and SAD amplifications. The nucleotide sequence of SAD is indicated in black. In FIG. 9 schematically shows the process of cutting the vector pET28 a (+) restriction endonucleases.

In FIG. 10 schematically illustrates a process for preparing an SAA PCR product using f-SA-XBA and f-SA-XNO oligonucleotides. The nucleotide sequence of SAA is indicated in black on the scheme. On the right in the diagram is the final amplification product - SAA.

In FIG. 11 schematically illustrates the ligation process of the pEP22a (+) / XbaI / XhoI construct and SAA amplifications. The nucleotide sequence of SAA is indicated in black.

In FIG. 12 schematically illustrates the ligation process of the pEP28a (+) - SAA / XbaI construct and nucleic acid sequence amplifiers encoding the TAT protein. The nucleotide sequence of SAA is indicated in black. The nucleotide sequence of the TAT peptide is indicated in gray.

A brief description of the drawings.

FIG. 1 Schematic representation of the synthesis of monovalent streptavidin with a TAT peptide in E. coli strain. At the beginning, the expression of the genes of the constructed plasmid DNA occurs, which provides the synthesis of mutant streptavidin monomers (Positions 1,2,3) linked to the PelB leader peptide (transporter protein in periplasm) and wild-type streptavidin monomers (position 4) linked to TAT- a peptide (a protein of the virus, human immunodiphosphitis) and an oligohistidine sequence. Then, tetramerisation of the protein monomers in the cytoplasm of the cell occurs (position 5) and transportation of this tetramer to the periplasm of the cell, where the PelB leader peptide is cleaved by cell proteases (position 6).

FIG. 2 Schematic representation of the synthesis of bivalent streptavidin with a TAT peptide in E. coli strain. Initially, the expression of the genes of the constructed plasmid DNA occurs, which provides the synthesis of mutant streptavidin monomers (Positions 1, 2) linked to the PelB leader peptide (transporter protein in periplasm) and wild-type streptavidin monomers (position 3, 4) linked to the TAT peptide (protein of the human immunodiphicitis virus) and oligohistidine sequence. Then, tetramerisation of the protein monomers in the cytoplasm of the cell occurs (position 5) and this tetramer is transported to the periplasm of the cell, where the PelB leader peptide is cleaved by the cell proteases (position 6).

FIG. 3 Schematic representation of the synthesis of tetravalent streptavidin with a TAT peptide in E. coli strain. Initially, the expression of the genes of the constructed plasmid DNA occurs, which provides the synthesis of mutant streptavidin monomers (Position 2) linked to the PelB leader peptide (transporter protein in periplasm) and wild-type streptavidin monomers (position 1, 3, 4) linked to the TAT peptide (protein of the human immunodiphicitis virus) and oligohistidine sequence. Then, tetramerisation of the protein monomers in the cytoplasm of the cell occurs (position 5) and transportation of this tetramer to the periplasm of the cell, where the PelB leader peptide is cleaved by cell proteases (position 6).

FIG. 4 Schematic representation of the synthesis of wild-type streptavidin homotetramer with a TAT peptide in E. coli strain. First, the expression of the genes of the constructed plasmid DNA occurs, which provides the synthesis of mutant streptavidin monomers (not shown in Fig.) Linked to the PelB leader peptide (transporter protein in periplasm) and wild-type streptavidin monomers (position 1, 2, 3, 4), linked to the TAT peptide (protein of the human immunodiphicitis virus) and oligohistidine sequence. Then, tetramerization of wild-type streptavidin monomers takes place in the cytoplasm of the cell (position 5). This tetramer does not include a mutant streptavidin that carries a PelB leader peptide, and therefore tetramers of this design will not be transported to the periplasm. Thus, it is possible in vivo to purify protein heterotetramers from the non-target streptavidin homotetramer.

FIG. 5 Schematic representation of the synthesis of homotetramers of mutant streptavidin in E. coli strain. First, the expression of the genes of the constructed plasmid DNA occurs, which provides the synthesis of mutant streptavidin monomers (Position 1, 2, 3, 4) linked to the PelB leader peptide (transporter protein in periplasm) and wild type streptavidin monomers (not shown in Fig.), linked to a TAT peptide (human immunodeficiency virus protein) and oligohistidine sequence. Then, tetramerisation of the mutant streptavidin monomers takes place in the cytoplasm of the cell (position 5) and this tetramer is transported to the periplasm of the cell, where the PelB leader peptide is cleaved by the cell proteases (position 6). This tetramer does not contain wild-type streptavidin bearing an oligohistidine sequence, and therefore tetramers of this design cannot be isolated from the periplasmic fraction on a nickel-agarose sorbent. Thus, it is possible to clean the heterotetramers of the protein from the non-target homotetramer of streptavidin.

The proposed method for the preparation of heterotetrameric streptavidin includes several stages:

- the creation of the plasmid pET22b + -SAD, providing the expression of mutant streptavidin linked to the pelB leader, transporting the heterotetrameric protein into the periplasm;

- the creation of the plasmid pET22a + -SAA-TAT, which provides the expression of wild-type streptavidin linked to the oligohistidine sequence and the TAT protein of human immunodeficiency virus;

- transformation of E. coli strain BL21 (DE3) with constructs pET22b + -SAD and pET22b + -SAA-TAT, followed by cultivation of this strain in LB medium;

- isolation of heterotetrameric streptavidin from the periplasmic space on a nickel-agarose sorbent;

- confirmation of the presence of protein in samples using electrophoretic separation of the protein under denaturing conditions; testing the ability of streptavidin to bind biotin using the method of immunoblotting.

Example 1. The creation of plasmids pET22b + -SAD

The original vector pET22b (+) [Novagen, pET System Manual, 11th Edition] containing the ampicillin resistance gene and the nucleotide sequence of the pe1B leader peptide [Novagen, pET System Manual, 11th Edition) was used to create a plasmid encoding the nucleotide sequence of mutant streptavidin ], necessary for the directed transport of protein into the periplasm. To generate the original vector pET22b (+), E. coli strain DH5 (α) was used. To 100 μl of competent E. coli DH5 (α) cells, 100 to 300 ng of pET22b (+) plasmid DNA was added. Then incubated in ice for 15 minutes. To effect heat shock, the cell suspension was incubated for 5 minutes at 42 ° C in a water bath, followed by incubation in ice for 2 minutes. 900 μl of LB medium was added to the suspension and incubated for 1 hour at 37 ° C. Cells were plated on plates containing agarized LB medium with ampicillin (100 μg / ml). The plates were incubated overnight at 37 °. E. coli DH5 (α) cells transformed with pET22b (+) were grown at 37 ° C under aeration, precipitated by centrifugation, and used to isolate plasmid DNA using the phenol-chloroform method. The supernatant was discarded, the cell residue was resuspended in 4 ml of medium and precipitated by centrifugation, then the supernatant was again discarded. 200 μl of solution I was added to each tube. Solution I has the following composition: 50 mM glucose, 25 mM TrisHCl pH 8.0, 10 mM EDTA pH 8.0. The mixture was resuspended on a vortex and 400 μl of solution II was added, with the following composition: 0.2N NaOH, 1% SDS. The resulting suspension was stirred by rapid inversion for 5 minutes and 300 μl of solution III was added (5M CH ₃ COOK, 11.5% CH ₃ COOH), inverted several times and incubated in ice for 5 minutes. Cells were pelleted by centrifugation and 600 μl of suspension were taken and transferred to pure eppendorfs and then 600 μl of isopropyl alcohol (-20 ° C) was added. Then the suspension was incubated at -20 ° C for 10 minutes and precipitated by centrifugation. The alcohol was drained and the precipitate was dried. Dissolve the precipitate in 200 μl of water. 250 μl of a phenol: chloroform mixture (1: 1) was added and mixed. The suspension was precipitated by centrifugation, the supernatant was collected and 250 μl of a mixture of chloroform: isoamyl alcohol (24: 1) was added and stirred. The suspension was precipitated by centrifugation. The resulting supernatant was collected, 2.5 volumes of 96% ethanol were added and incubated at -20 ° C for 30 minutes. Then it was precipitated by centrifugation, the alcohol was drained and 1 ml of 70% ethyl alcohol was added. Then the mixture was precipitated by centrifugation, the alcohol was drained. The precipitate was dried until the smell of alcohol disappeared. The precipitate was dissolved in 200 μl of water. The resulting plasmid DNA was separated on a 0.8% agarose gel. The composition of the gel: 0.8% agarose, 0.5x TBE buffer, 1 μg / ml ethidium bromide. Composition sample buffer: 30% glycerol (v / v), 0.025% xylencyanol (w / v), 0.025% bromophenol blue (w / v). Electrode buffer: 0.5x TBE buffer Composition of 10x TBE buffer: 890 mM Tris, 890 mM boric acid (pH 8.0), 20M EDTA. To prepare the gel, the mixture of components was heated until the agarose was completely dissolved, then it was cooled to 50 ° С, ethidium bromide was added to a concentration of 0.5 mg / ml and it was poured onto a horizontal platform measuring 80 × 110 mm. The gel thickness was 5 mm. The electric field strength was 5 V / cm. Commercial markers 250-10000 bp were used as markers. ("Medigen"). The plasmid was purified from agarose gel using a special Promega kit (WizardSV Gel and PCR Clean-Up System, USA). - delete everything

To insert the nucleotide sequence of mutant streptavidin (SAD) into the original plasmid, “sticky ends” obtained by processing the restriction endonuclease vector NcoI and XhoI were used (Fig. 6). By mutant streptavidin is meant streptavidin, unable to bind biotin.

, 1 час):The restriction reaction mixture had the following composition (V = 60 μl,

, 1 hour):

pET22b (+): 22 μl

NcoI 2.5 μl, XhoI 2.5 μl,

10 ^X buffer 6 μl,

H ₂ About 27 μl

After the hydrolysis procedure, the resulting linear vector was subjected to electrophoretic separation in 0.8% agarose gel using the protocol indicated above. The plasmid was purified from agarose gel using a special Promega kit (WizardSV Gel and PCR Clean-Up System, USA).

To generate the required amount of the SAD nucleotide sequence, PCR was performed using specially selected oligonucleotides.

A plasmid containing the nucleotide sequence of a mutant streptavidin [Biochem Biophys Res Commun. 2015 Aug 7; 463 (4): 1059-1063]. To obtain the nucleotide sequence, we selected special oligonucleotides:

f-SA-NCO: 5'- ctg ccatgg ctgaagctggtatcacc is an oligonucleotide with a restriction site for NcoI and a sequence complementary to the streptavidin gene

r-SA-stop-XHO: 5'- ctg ctcgag tcattoggaagcagcggacggtt - with the XhoI restriction site, the stop codon in front of the oligo-histidine sequence and the sequence of the complementary sequence of the streptavidin gene.

30 циклов, 1 минута 72°С (достройка), 5 минут 72°С.The polymerase chain reaction (PCR) was carried out in the reaction mixture with a final volume of 30 μl. Composition of the reaction mixture: Oligonucleotides (direct and reverse) of 10 pMol, Taq polymerase buffer x1, (dATP, dTTP, dGTP, dCTP) 6 nMol, MgCl ₂ 50 nMol, Taq polymerase 1 unit. PCR was performed in 0.5 ml microcentrifuge tubes on a Tertsik apparatus (Russia) with a set of programs determining the temperature regime of PCR. Amplification of nucleotide sequences was carried out by PCR. PCR temperature profile: 5 minutes 95 ° C, 1 minute 95 ° C (denaturation), 1 minute

30 cycles, 1 minute 72 ° C (completion), 5 minutes 72 ° C.

The SAD product preparation scheme is shown in FIG. 7.

, 1 час):The insertion of the nucleotide sequence of SAD in the plasmid pET22b (+) was performed as follows. The obtained amplification products of the nucleotide sequence of SAD were processed with restriction endonucleases XhoI and NcoI. The composition of the reaction mixture for NcoI and XhoI (V = 60 μl,

, 1 hour):

SAD nucleotide sequence: 35 μl

NcoI 2.5 μl, XhoI 2.5 μl,

10 ^X buffer 6 μl,

H ₂ O 16 μl

Thus, we prepared the plasmid pET22b (+) / NcoI / XhoI and the amplification products of the SAD nucleotide sequence with identical sticky ends. The products of restriction of the nucleotide sequence of SAD were mixed with the prepared vector and then were marked. The molar ratio of plasmid and PCR product was taken 1/20. For ligation used a ligase company "Fermentas" (Lithuania). The composition of the reaction mixture (sample volume was 10 μl.): SAD nucleotide sequence 65 ng (4 μl), Ligase 1 μl, Ligase buffer 1 μl, Plasmid 40 ng (4 μl). The lettering was carried out for 16 hours at a temperature of + 4 ° C. As a result, we obtained a genetic construct called pEP22b (+) - SAD, which encodes an SAD protein with a PelB leader peptide. The circuit for creating these structures is shown in FIG. 8.

The selection of colonies containing plasmids with the insertion of the nucleotide sequence of SAD was performed as follows. The resulting ligase mixture was transformed competent cells of strain DH5 (α) E. coli. From 100 to 300 ng of pET22b (+) - SAD plasmid DNA was added to 100 μl of competent E. coli DH5 (α) cells and further transformation was carried out using the same method described above. Transformants were plated on a Petri dish with agarized LB medium, which included the antibiotic ampicillin. Thus, ampicillin-resistant cells containing plasmids, ligation products, grew on a selective medium.

E. coli DH5 (α) cells transformed with pET22b (+) - SAD were grown at 37 ° C under aeration, pelleted by centrifugation at 4000 rpm in a K23 centrifuge at 4 ° C for 30 minutes, and used to isolate plasmid DNA the method described in detail above in the text.

Several colonies were selected to verify insertion by PCR. The nucleotide sequence of the target genetic construct was further verified by Sanger sequencing [J. Mol. Biol. 1975. V. 94. P. 441-446].

Example 2. The creation of the plasmid pET28a + -SAA-TAT

To create a plasmid encoding the nucleotide sequence of wild-type streptavidin, we used the original vector pET28a (+) [Novagen, pET System Manual, 11th Edition] containing the kanamycin and oligohistidine resistance gene necessary for subsequent purification of the target protein on a nickel-agarose sorbent . To produce the initial vector pET28a (+), E. coli strain DH5 (α) was used. From 100 to 300 ng of pET28a (+) plasmid DNA was added to 100 μl of competent E. coli DH5 (α) cells, and then transformation was carried out using the same method described above. E. coli DH5 (α) cells transformed with pET28a (+) were grown at 37 ° C under aeration, precipitated by centrifugation, and used to isolate plasmid DNA using the method described in detail above. The resulting plasmid DNA was separated on a 0.8% agarose gel using the protocol indicated above. The agarose gel plasmid was purified using a Promega kit (WizardSV Gel and PCR Clean-Up System, USA).

To insert fragments of the nucleotide sequence of wild-type streptavidin (SAA) into the original plasmid, “sticky ends” obtained by treating the vector with XbaI and XhoI restriction endonucleases were used (Fig. 9).

The restriction reaction mixture had the following composition: the composition of the reaction mixture for XbaI and XhoI (V = 60 μl, t0 = 370C, 1 hour):

pET28a (+): 22 μl

XbaI 2.5 μl,

XhoI 2.5 μl,

10 ^X buffer 6 μl,

H ₂ About 27 μl

After the hydrolysis procedure, the resulting linear vector was subjected to electrophoretic separation in 0.8% agarose gel using the protocol indicated above in the text. The agarose gel plasmid was purified using a Promega kit (WizardSV Gel and PCR Clean-Up System, USA).

To generate the required amount of the SAA nucleotide sequence, PCR was performed using specially selected oligonucleotides.

A plasmid containing the nucleotide sequence of wild-type streptavidin was used as a matrix for generating the nucleotide sequence [J Chromatogr A. 1994 Aug 5; 676 (2): 337-45]. To obtain the nucleotide sequence of SAA, we selected special oligonucleotides:

f-SA-XBA: 5'-ctgtctagactgaagctggtatcacc is an oligonucleotide with a restriction site for XbaI and a sequence complementary to the streptavidin gene

r-SA-XHO: 5'- ctg ctcgag ggaagcagcggacggtt is an oligonucleotide with a restriction site for XhoI, a sequence complementary to the streptavidin gene

The SAA product preparation scheme is shown in FIG. 10.

, 1 час):Insertion of the nucleotide sequence of SAA into plasmid pET28a (+) was carried out as follows. The resulting SAA amplification products were pretreated with XhoI and XbaI restriction endonucleases. The composition of the reaction mixture for XbaI and XhoI (V = 60 μl,

, 1 hour):

SAA nucleotide sequence: 35 μl

XbaI 2.5 μl, XhoI 2.5 μl,

10 ^X buffer 6 μl,

H ₂ O 16 μl

Thus, we prepared the plasmid pET28a (+) / XbaI / XhoI and amplification products of the nucleotide sequence of SAA with identical sticky ends. The restriction products of the nucleotide sequence of SAA were mixed with the prepared vector and then ligated. The molar ratio of plasmid and PCR product was taken 1/20. For ligation used a ligase company "Fermentas" (Lithuania). The composition of the reaction mixture (sample volume was 10 μl): nucleotide sequence SAA 65 ng (4 μl), Ligase 1 μl, Ligase buffer 1 μl, Plasmid 40 ng (4 μl). Ligation was carried out for 16 hours at a temperature of + 4 ° C. As a result, we obtained a genetic construct, which we conventionally called pEP28a (+) - SAA, expressing the SAA gene linked to the oligohistidine sequence. The circuit for creating these structures is shown in FIG. eleven.

The selection of colonies containing plasmids with the insertion of the nucleotide sequence of SAA was carried out in the following way. The resulting ligase mixture was transformed competent cells of strain DH5 (α) E. coli. From 100 to 300 ng of pET22b (+) - SAA plasmid DNA was added to 100 μl of competent E. coli DH5 (α) cells and further transformation was performed using the same method described above. Transformants were plated on a Petri dish with agarized LB medium, which included the antibiotic kanamycin. Thus, on a selective medium, kanamycin-resistant cells containing plasmids — ligation products — grew.

E. coli DH5 (α) cells transformed with pET28a (+) - SAA were grown at 37 ° C under aeration, pelleted by centrifugation at 4000 rpm in a K23 centrifuge at 4 ° C for 30 minutes, and used to isolate plasmid DNA the method described in detail above in the text.

Several colonies were selected to verify insertion by PCR. The nucleotide sequence of the target genetic construct was further verified by Sanger sequencing [J. Mol. Biol. 1975. V. 94. P. 441-446].

The insertion of the nucleotide sequence encoding the TAT peptide into the plasmid pEP28a (+) - SAA was performed as follows. The previously obtained vector was prepared for insertion of the nucleotide sequence of the TAT peptide. For this, the vector was subjected to restriction at the XbaI site. The composition of the reaction mixture for XbaI (V = 60 μl,

t0 = 370C, 1 hour):

pET28a (+) - SAA: 22 μl

XbaI 4 μl,

10 ^X buffer 6 μl,

H ₂ O 28 μl

After the hydrolysis procedure, the resulting linear vector was subjected to electrophoretic separation in 0.8% agarose gel using the protocol indicated above. The agarose gel plasmid was purified using a Promega kit (WizardSV Gel and PCR Clean-Up System, USA) ..

The number of nucleotide sequences of the TAT peptide required for insertion into the plasmid was obtained using the method described in detail in the article [J Drug Target. 2012 Sep; 20 (8): 678-690]. Amplification products of the nucleotide sequence of the TAT peptide were pretreated with Xbal restriction endonuclease.

The reaction mixture for XbaI_ (V = 60 μl, t0 = 370C, 1 hour): nucleotide sequence of TAT: 35 μl

XbaI 4 μl,

10 ^X buffer 6 μl,

H ₂ About 15 μl

The restriction products of the nucleotide sequence of the TAT peptide were mixed with the prepared vector and then ligated. The molar ratio of plasmid and PCR product was taken 1/100. For ligation used a ligase company "Fermentas" (Lithuania). The composition of the reaction mixture (sample volume was 10 μl): nucleotide sequence of the TAT peptide 65 ng (4 μl), Ligase 1 μl, Ligase buffer 1 μl, Plasmid 40 ng (4 μl). Ligation was carried out for 16 hours at a temperature of + 4 ° C. As a result, a genetic construct was obtained that we conventionally called pEP28a (+) - SAA-TAT, expressing the nucleotide sequence of SAA linked to the nucleotide sequence of the TAT peptide. The circuit for creating these structures is shown in FIG. 12.

The resulting ligase mixture was transformed competent cells of strain DH5 (α) E. coli. From 100 to 300 ng of pET22b (+) - SAA-TAT plasmid DNA was added to 100 μl of competent E. coli DH5 (α) cells and further transformation was performed using the same method described above. Transformants were plated on a Petri dish with agarized LB medium, which included the antibiotic kanamycin. Thus, on a selective medium, kanamycin-resistant cells containing plasmids — ligation products — grew.

E. coli DH5 (α) cells transformed with pET28a (+) - SAA-TAT were grown at 37 ° C under aeration, pelleted by centrifugation, and used to isolate the plasmid DNA described above.

Several colonies were selected to verify insertion by PCR. The nucleotide sequence of the target genetic construct was verified by Sanger sequencing [J. Mol Biol. 1975. V. 94. P. 441-446].

Example 3. Transformation of E. coli strain BL21 (DE3) constructs pET22b + -SAD and pET22b + -SAA-TAT

The resulting constructs pET22b (+) -SAA-TAT and pET22b + -SAD were used to transform E. coli BL21 (DE3) cells. From 100 ng of pET22b (+) -SAA-TAT and pET22b (+) SAD plasmid DNA was added to 100 μl of competent E. coli BL21 cells (DE3). Then incubated in ice for 15 minutes. To effect heat shock, the cell suspension was incubated for 5 minutes at 42 ° C in a water bath, followed by incubation in ice for 2 minutes. 900 μl of LB medium was added and incubated for 1 hour at 37 ° C. Cells were plated on plates containing agarized LB medium with ampicillin (100 μg / ml) and kanamycin (50 μg / ml). The plates were incubated overnight at 37 °.

Example 4. Cultivation of E. coli strain BL21 (DE3) / pET28a (+) -SAA-TAT / pET22b + -SAD.

The resulting E. coli BL27 (DE3) / pET28a (+) -SAA-TAT / pET22b + -SAD transformants were seeded in 10 ml of LB medium supplemented with ampicillin (100 μg / ml) and kanamycin (50 μg / ml) grown overnight at 37 ° C with constant swaying. In the morning, the inoculum was poured into 500 ml of LB medium. To select cells transformed with both plasmids, the antibiotic ampicillin and kanamycin was added (calculated as 100 μg and 50 μg, respectively, per 1 ml of medium). Cells were grown at 37 ° C under aeration to a density of A600 = 0.6-1. Protein synthesis was induced by the addition of IPTG (0.5 mmol / L), cultivation was continued overnight at 26 ° C.

Example 5. Isolation of protein from the periplasmic fraction of E. coli BL21 (DE3).

After separating the bacterial cells by centrifugation for 15 minutes at 10,000 g, the periplasmic fraction was obtained by the osmotic shock method [Current Protocols in Molecular Biology. 1989. John Wiley & Sons, New York]. The streptavidin heterotetramers were affinity purified on a metal-chelated nickel-agarose sorbent (Sigma). For this, phenylmethylsulfonyl fluoride and imidazole were added to the obtained bacterial periplasm content (final concentration of 1 mM and 10 tM, respectively). The solution was filtered through a metal-chelate sorbent. Ballast proteins were washed with a 0.5 m NaCl solution in 0.1 M K-phosphate buffer, pH = 8.0, containing 20 mM imidazole. Next, the protein was eluted with 0.15 M NaCl, pH = 7.4, containing 200 mM imidazole.

Example 6. Electrophoretic separation of protein in SDS-electrophoresis.

Protein-containing fractions were analyzed by electrophoretic separation. Electrophoretic separation of proteins was carried out in 12% polyacrylamide gel 9 * 9 cm with a voltage gradient of 20 V / cm in a separation gel of the following composition: 12% acrylamide / methylene bisacrylamide 29: 1, 37 mm Tris-HCl, pH = 9.2, 0.01 % ammonium persulfate, 0.001% tetramethylethylenediamine using an electrode buffer containing 0.125 M Tris-HCl, pH = 6.8.

Samples were mixed in a 4: 1 ratio with a buffer consisting of 0.3 M TrisHCl, pH 6.8, 30% glycerol, and 0.025%) bromophenol blue. To conduct electrophoretic analysis of proteins in PAGE under denaturing conditions, DSD was added to the composition of gels, electrode buffer and sample buffer to a final concentration of 0.1%. 5% mercaptoethanol was added to the sample buffer, and the protein mixture was heated for 10 minutes at 100 ° C.

For electrophoretic separation of proteins, an electrophoretic installation with an Elf-4 power supply was used.

The presence of protein in the samples was confirmed using the following experiment. Samples with preliminary boiling at 100 ° С and with the addition of mercaptoethanol and a sample without preliminary boiling were applied to neighboring wells on the gel. The streptavinide protein tetramer has a molar mass of about 60 kDa and gives a bend on the gel in the appropriate place for this molecular weight. Upon boiling, the tetramer structure of this protein is destroyed, and it decomposes into monomers giving bends on a gel in the region of molecular masses equal to 15 kDa. The presence of bends in the indicated areas of molecular masses simultaneously, namely, in the region of 60 kDa in the case of applying samples without boiling and in the region of 15 kDa in the case of applying samples with boiling, indicates the presence of streptavidin protein in the sample. Thus, using this experiment, it was possible to confirm the presence of streptavidin protein in samples isolated from the periplasmic fraction.

Example 9. Verification of protein activity by immunoblotting.

To test the obtained heterotetrameric streptavidin for the ability to bind biotin, the method of immunoblotting was used. On a nitrocellulose filter 1 × 1 cm or 2 × 2.5 cm, 1 μl of streptavidin heterotetromeres was applied. "Coloring" nitrocellulose filter was carried out using the following solutions:

Solution I: 3% skim milk (Sigma, USA) per

FBI (filtered).

Solution II: Diluted 3 times solution I.

Solution III: 6 mg of 4-chloro-1-naphthol, 2 ml of 96% ethanol, up to 12 ml of PBS, 6 μl of 3033% hydrogen peroxide.

The nitrocellulose filter was incubated in solution I for 30 minutes with constant shaking at room temperature. Then they were incubated with biotinylated peroxidase in solution I for 1 hour while shaking at room temperature and washed 3 times for 5 minutes with solution II.

Then they were incubated for 1 hour with shaking at room temperature with a second antibody streptavidin conjugated to horseradish peroxidase (ICN, USA). The required number of second antibodies was calculated based on the protocol of the manufacturer. The nitrocellulose filter was washed three times for 5 minutes with the FBI and stained with solution III.

Using this experiment, it was possible to confirm the presence of active streptavidin in the samples.

Claims (18)

1. A method of producing a heterotetrameric recombinant streptavidin from E. coli periplasm, comprising the creation of two plasmids for the expression of streptavidins, transformation of the obtained E. coli cell plasmids, culturing cells in a culture medium, inducing the synthesis of streptavidins with isopropyl-β-D-thiogalactopyranoside, isolation periplasmic space and purification of heterotetrameric streptavidin on an affinity sorbent, characterized in that as the first plasmid, a plasmid is created on the basis of the initial vector, which expresses wild-type streptavidin linked to an affinity tag and has an antibiotic resistance gene of the aminoglycoside group, as a second plasmid, a plasmid is created on the basis of the initial vector, which provides expression of mutant streptavidin linked to the pelB leader peptide and incapable of binding biotin, which has the penicillin antibiotic resistance gene, carry out the transformation of the same E. coli cells with the two indicated plasmids, followed by culturing these transformed cells in a culture medium containing both an antibiotic of the penicillin series and an antibiotic of the aminoglycoside group, or characterized in that as the first plasmid, a plasmid is created on the basis of the initial vector, which provides expression of mutant streptavidin linked to an affinity tag and is not capable of binding biotin, which has an antibiotic resistance gene of the aminoglycoside group, as a second plasmid, a plasmid is created on the basis of the initial vector, which expresses wild-type streptavidin linked to a pelB leader peptide having the penicillin antibiotic resistance gene, carry out the transformation of the same E. coli cells with the two indicated plasmids, followed by culturing these transformed cells in a culture medium containing both an antibiotic of the penicillin series and an antibiotic of the aminoglycoside group. 2. The method according to p. 1, characterized in that nickelagarose is used as the affinity sorbent. 3. The method according to p. 1, characterized in that ampicillin is used as an antibiotic of the aminoglycoside group. 4. The method according to p. 1, characterized in that kanamycin is used as an antibiotic of the penicillin series. 5. The method according to p. 1, characterized in that the vector pET28a + can be used as a vector for the first plasmid. 6. The method according to p. 1, characterized in that the vector pET22b + can be used as a vector for the second plasmid. 7. The method according to p. 1, characterized in that the strain E. coli BL21 (DE3) is used as a strain for transforming E. coli cells. 8. The method according to p. 1, characterized in that the mutant streptavidin, not capable of binding biotin, or wild-type streptavidin is linked to a fusion peptide. 9. The method according to p. 8, characterized in that the TAT peptide is used as the fusion peptide. 10. The method according to p. 1, characterized in that as an affinity tag using the oligohistidine sequence.

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