RU2572343C2 - RECOMBINANT DNA pA4, RECOMBINANT DNA pQE 30-pA4, PROVIDING OBTAINING POLYPEPTIDE A4, Esherichia coli STRAIN M 15-A4, TRANSFORMED WITH RECOMBINANT PLASMID DNA pQE 30-pA4, AND EXPRESSING RECOMBINANT POLYPEPTIDE A4, RECOMBINANT POLYPEPTIDE A4, POSSESSING ABILITY TO SELECTIVELY BIND HSA, AFFINE SORBENTS (VERSIONS) AND METHODS FOR HSA AND IgG REMOVAL FROM BLOOD SERUM (VERSIONS) - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: inventions relate to field of molecular biology and deal with recombinant DNA pA4, recombinant plasmid DNA pQE 30-A4, Esherichia coli strain M15-A4, recombinant polypeptide A4, possessing ability to selectively bind human serum albumin (HSA), affine sorbent, containing such polypeptide, affine combined sorbent and methods for successive removal of HSA and IgG from blood serum. Characterised affine combines sorbent is obtained by mixing affine sorbent, containing said recombinant polypeptide A4, and analogous affine sorbent, in which known IgG-binding polypeptide is used as ligand.

EFFECT: claimed inventions can be used in medical practice to deplete blood serum of two proteins, albumin (HSA) and immunoglobulin G (IgG), present in it in high concentrations Removal of two major proteins from blood serum will make it possible to identify other proteins, present in serum in lower concentrations.

8 cl, 9 dwg, 7 ex, 1 tbl

Description

The invention relates to the field of molecular biology and proteomics and can be used in medical practice as an available reagent for the release of blood serum from two proteins, albumin (HSA) and immunoglobulin G (IgG), which are in it in high concentrations. Removal of two major proteins from blood serum will determine other proteins present in serum at lower concentrations.

In recent years, one of the promising directions in creating diagnostic methods for various diseases is the use of proteomic technologies, the scope of which is closely related to the search for diagnostic markers of diseases. The main idea of such a targeted “marker” diagnosis is the identification of certain molecules or their complexes that are present only in the affected tissues or cells and / or are released into the external environment or internal environment of the body.

Objects of research can be biological fluids (plasma or blood serum, cerebrospinal fluid, synovial, amniotic, bronchoalveolar fluids, urine), cells and biopsy samples of human tissues, as well as proteomes of microorganisms - pathogens [Arevalo-Ferro C. et al., Environmental Microbiology 5 (12): 1350-1369, 2003].

Blood serum (plasma) is readily available and most convenient for diagnostic use. Serum is a rich source of biochemical products that can act as indicators of the physiological and clinical condition of the patient. For example, levels of hormones, cholesterol, enzymes, or other proteins can provide information on cardiovascular disease or nutritional status, viral disease, or cancer. Therefore, a lot of effort is being made to search for new biomarkers for the diagnosis of various diseases, more and more analytical methods are being used to study the profile of the content of human serum proteins. By methods of proteomic analysis, it is possible to analyze up to 10,000 individual proteins in one sample and record changes in their concentrations, which allows the diagnosis and monitoring of the course of the disease.

The main task of proteomics is to identify the mechanism of interaction of a huge number of proteins and peptides in one organism. Very often there is a close relationship between changes in the protein composition and the painful condition of a person. Therefore, new data in proteomics are used to quickly develop new medicinal properties and the latest methods of treating diseases that medicine has struggled with for centuries. Today, most pharmacological agents affect proteins. Proteomics with its systematic approach can help to identify and assess the importance of the appearance of new proteins much more efficiently, which, in turn, will accelerate the development of new diagnostic tests and therapeutic agents. Serum proteome can be analyzed by various methods, among which one of the most effective is the method of two-dimensional electrophoresis (2-DE). The resolution of this method is so great that it allows you to separate two almost identical proteins that differ in one charged amino acid. As a result of 2-DE, a two-dimensional “protein map” is obtained. To identify biomarkers, “proteomic maps” of samples are compared in a pathological condition and normal, revealing proteins whose expression level changes with the development of pathology. Next, these proteins are excised from the gel and hydrolyzed by any endopeptidase, for example, trypsin (trypsinolysis) to obtain short (in an average of 10-15 amino acid residues) of peptides that are identified in the next step of the analysis. MALDI-TOF mass spectrometers make it possible to conduct direct mass spectrometric analysis of blood serum proteins (direct protein profiling) and obtain unique mass spectra with high accuracy and resolution that characterize the object under investigation as “fingerprints” [M.A. Stowers et. al., Rapid Commun. Mass Spectrom. 14: 829-833, 2000]. They allow you to quickly analyze a large number of samples, require a relatively small amount of biological material, have high sensitivity and good resolution for low molecular weight proteins and peptides, which makes them especially promising for the search for biomarkers. The next step is to determine the amino acid sequence of the peptide fragments. There are databases containing the amino acid sequences of proteins, and by querying the database for the known peptide sequence, a protein is identified [Govorun VM, Archakov AI, Biochemistry, 67, 10: 1341-1359, 2002].

However, the use of proteomics technologies to search for potential diagnostic and prognostic biomarkers in the blood of patients is limited by the presence of HSA and IgG in high concentrations. Their removal from blood serum should facilitate the detection of other proteins located both in the high molecular weight region (70 kDa) shielded by HSA and in the region of 30-50 kDa, where a wide IgG band hides proteins migrating in this region. It is the ratio of minor components in plasma that is of greatest interest to proteomic studies.

In most cases, Cibacron blue ligand is used to remove HSA, which, having strong nonspecific binding, impedes analysis by two-dimensional electrophoresis [Laura F. Steel et. al., Proteomics 3: 601-609, 2003]. In addition to HSA, other whey proteins also bind to this ligand. Therefore, to increase the specificity of the reaction, sorbents with antibodies to HSA attached to them were used, which turned out to be disadvantageous due to the low capacity of the sorbent and high cost [V. Polaskova, Electrophoresis, 31: 471-482, 2010]. To remove HSA from serum, antibodies to HSA are sometimes used, and IgG is used by monofunctional G protein. Aurum ™ Affi-Gel Blue kit (Bio-Rad), the ProteoPrep immunoaffinity albumin and IgG depletion kit (Sigma), the Multiple Affinity Removal System (MARS column Agilent Technologies) have recently appeared. These kits are based on the use of affinity columns to which antibodies to HSA and IgG immunoglobulin are attached. Work with these kits showed that in addition to HSA and IgG, many other serum proteins are also removed, which indicates the lack of specificity of these kits [Elisa Bellei, Stefania Bergamini, Amino Acids, 40: 145-156, 2011]. All existing techniques are based on the antigen-antibody reaction, which can produce non-specific reactions. Therefore, these methods are not effective enough. Some foreign authors have tried to create a variant of the recombinant HSA-binding polypeptide from group G streptococcus isolated from humans, which would have high affinity for HSA [A. Jonsson et al, Engeneering, Design Selection, 21, 8: 515-527, 2008]. A variant of the recombinant HSA-binding polypeptide was obtained, which had a very high affinity for HSA with Co, of the order of 10 ^-15 . However, these works were unsuccessful, since the addition of this polypeptide to the sorbent for affinity chromatography did not increase the capacity of this sorbent, which made it impossible to use it for complete release of serum from HSA [A. Jonsson et al, Engeneering, Design Selection, 21, 8: 515-527, 2008].

The use of a recombinant bifunctional protein G, which simultaneously binds HSA and IgG, to remove CSA and IgG from blood serum is described [E.A. Bormotova, T.V. Gupalova. Biotechnology, No. 2: 49-55, 2009].

This technical solution was made as a prototype of the claimed group of inventions.

This method has drawbacks due to the use of a bifunctional G protein, which binds HSA with lower efficiency than IgG. 90% CSA removal occurred when only 10 μl of serum was applied to the column, which is clearly not enough.

The objective of the invention is to solve an urgent problem - the creation of a new method for the effective removal of HSA and IgG from blood serum, based on the interaction of protein-protein using recombinant receptor HSA and IgG-binding polypeptides obtained from streptococci, the purification quality of which provides almost 100% sorbent specificity.

The claimed group of inventions, united by a single inventive concept, which includes the following objects:

1. Recombinant DNA (designated as pA4) obtained by polymerase chain reaction (PNR) using the chromosomal DNA of group D streptococcus strain DG13, unique primers PA1 and PA2 and subsequent cloning using the expression plasmid pQE-30 (The QIAexpress System, Qiagen , USA).

2. Recombinant plasmid DNA pQE 30-pA4, carrying recombinant pA4 DNA.

3. The producer strain E. coli M15-A4, allowing under certain conditions to express recombinant A4 polypeptide.

4. Recombinant A4 polypeptide containing a sequence of two HSA-binding GA modules and region W, having a high ability to bind HSA and having a lower molecular weight than the A2 polypeptide [T.V. Gupalova et al., Biotechnology, No. 3: 75-84, 2012].

5. An affinity sorbent, in which an HSA-binding polypeptide is attached to a matrix of cyanbrom-activated 4B Sepharose.

6. The affinity combined sorbent, consisting simultaneously of two sorbents: Sepharose 4B-IgG-binding polypeptide and the sorbent according to paragraph 5.

7. A method for sequentially removing HSA and IgG from blood serum by affinity chromatography, characterized in that the 4B-IgG-binding polypeptide and sorbent according to claim 5 are used as affinity carriers in series-connected columns.

8. The method of simultaneous removal of HSA and IgG from blood serum, characterized in that the affinity combined sorbent according to claim 6 is used as the sorbent.

The technical essence and the technical result achieved by using the claimed group of inventions are as follows.

HSA and IgG are the most common plasma proteins in humans and mammals.

In recent decades, bacteria proteins with receptor activity against HSA and IgG have been identified and studied.

Streptococci (Streptococcus) of groups A, C and G express surface proteins that bind HSA [Myhre E. and Kronvall G. Infect. Immun. 27: 6-14, 1980; Wideback K. et. al., Acta Pathol. Microbiol. Immunol. Scand. Sect. B. 91: 373-382, 1983].

Among such surface proteins of group G streptococci, protein G is of interest, the full-sized molecule of which has the ability to bind human IgG and various mammals, as well as human and animal HSA [Bjorck L. et. al. Mol. ImmunoL 24: 1113-1122, 1987].

A recombinant G protein that simultaneously binds HSA and IgG, a bifunctional G protein and a monofunctional G protein that binds only IgG, that is, an IgG-binding polypeptide [Gupalova T.V., were obtained from a group G, G148 streptococcus strain isolated from humans. , Bulletin of the Academy of Medical Sciences, 3: 44-49, 1996].

From the Finegoldia magnus strain, a PAB protein binding to HSA was isolated [de Chateau M., Bjorck L., J. of Biol. Chem. 269: 12147-12151, 1994]. Analysis of its amino acid sequence revealed a domain of 45 amino acid residues responsible for binding to HSA.

It has been suggested that this domain has been moved from streptococci of groups C and G to the HSA-binding domain of Finegoldia magna strain. [Johansson M.U., Nilsson H. et al, J. Mol. BioL, 2002; 316: 1083-1099]. Such interspecific exchange was structurally defined as a mobile module and designated as GA module (G related albumin-binding domain).

The GA module sequence has homology to the HSA-binding region of protein G [B. Akerstrom et al., J of Biol. Chem. 262: 13388-13391, 1987;

Sjobring U. et. al., J. Immun. 140: 1595-1599, 1988; Nygren P. et. al., J. Mol. Recognit. 1: 69-74, 1988].

Unlike RAV protein, which does not have homologous repeats, protein G isolated from strain G 148 contains three homologous repeating sequences, i.e. three GA modules.

From a strain of Streptococcus canis DG12 isolated from cow's milk, an HSA-binding protein was obtained that contains two GA modules. It does not bind IgG, but in its ability to bind, HSA is superior to protein G [Sjobring U., Infection and Immun. 60: 3601-3608, 1992]. This property makes it attractive for practical use.

The study of the structure of IgG and HSA-binding proteins is of great importance for the technology of creating protein reagents relevant in immunochemistry, proteomics, biotechnology and clinical diagnostics.

The use of recombinant polypeptides allows you to: eliminate the time-consuming process of preparing specific antibodies to HSA or IgG (for which laboratory animals are used), avoid non-specific "background" reactions often found in immunology, and standardize the HSA or IgG binding polypeptides used.

The recombinant HSA receptor was first obtained by Swedish authors by cloning a fragment of the HSA receptor gene in a non-pathogenic host [Nygren P.-A. et al., J. Mol. Recognition 1: 69-74, 1988]. In this work, to clone the AB region (the region responsible for HSA binding), the plasmid pSPG2 was restricted with the EcoRI enzyme, followed by treatment with the Klenov fragment, addition of the synthetic SalI linker and ligation. By restriction with SalI and PstI, a 640 bp fragment was obtained, which was isolated from agarose and inserted between the same sites in the pEML8 vector. A gene fragment was excised from the obtained plasmid using EcoRI and HindIII sites and inserted into the pEG plasmid, the construction of which is described by Eliasson [Eliasson M. et al .; J. Biol. Chem. 263: 4323-4327, 1988].

As a result of this modification, the plasmid pB ₁ B ₂ was obtained, which is responsible for HSA-binding activity. A protein was isolated from a subclone producing an HSA-binding protein. It was purified by affinity chromatography. This protein was heterogeneous with the degradation of the purified product. The reason for the heterogeneity, obviously, was a rather complicated way of its genetic construction. Due to its shortcomings, this protein does not find practical application.

In 1996, a gene fragment encoding the third GA module of protein G of strain G148 was cloned. The obtained protein G148-GA3 was used to study the structure of the HSA-binding module [Kraulis P. et al., FEBS Lett. 378: 190-194, 1996], i.e. for research purposes only.

In 1992, a gene fragment encoding an HSA-binding fragment was cloned from a group G streptococcus of animal origin, strain DG 12, using the plasmid vector pKK233-2 and its nucleotide sequence was determined [Sjobring U. Infect, and Immun. 60: 3601-3608, 1992].

Recombinant HSA-binding receptor polypeptides A1 and A2 were obtained. Polypeptide A1 was obtained from group G streptococcus, strain G148 isolated from humans, in the Department of Molecular Microbiology in 1996 [S. Orlova et al. Biotechnology. 8: 13-21, 1996]. The strain producing the A1 polypeptide was donated to the collection of NIIEM SZO RAMP, No. 357.

A feature of this polypeptide is that it contains an HSA-binding region spanning three GA modules. A fragment of a gene that encodes A1 polypeptide has been studied. The study of this receptor polypeptide proved its ability to bind exclusively to the main human plasma protein - HSA. This property of the polypeptide was used in diagnostic test systems, allowing to determine the concentration of HSA in various biological fluids, in particular in urine. However, when isolating and purifying the recombinant A1 polypeptide, the yield of the active reagent in sufficient quantities was difficult, which led to the need to create a new HSA-binding drug with greater activity than A1 protein G polypeptide.

Polypeptide A2 was obtained from strain DG 13 of group G streptococcus isolated from cow's milk [Gupalova T.V. and other Biotechnology, No. 3: 75-84, 2012]. The strain producing the A2 polypeptide is deposited in the collection of NIIEM SZO RAMS, No. 593. It was shown that the A2 polypeptide has a greater HSA-binding activity compared to the A1 polypeptide from the group G streptococcus strain of human origin. A producer strain E. coli M 15-A2 was created, which allows one to obtain the A2 polypeptide with HSA-binding activity in high yield. The A2 polypeptide was used in the XRD method to determine microalbuminuria. It is characterized by a sequence of 346 amino acids in which the first 13 amino acids encoded by the sequence of plasmid pQE 32 are covalently linked to the subsequent 333 amino acids encoded by the sequence of the chromosomal DNA fragment of strain DG 13 of streptococcus group G and identical to the sequence of HSA-binding protein from strain DG 12 of streptococcus group G [Sjobring U. Infect, and Immun. 60: 3601-3608, 1992]. This sequence consists of 11 amino acids that are compatible with the last part of the leader peptide, i.e. it contains a series of hydrophobic residues with a small uncharged amino acid at the end. Then comes the unique sequence of 55 amino acids, designated N, followed by two regions designated E1 (28 amino acids) and E2 (29 amino acids). They are followed by sequences of two GA modules. In the following sequence, designated W, a sequence of four amino acids (LysProGluVal) was found, repeating 12 times.

The molecular weight of the HSA-binding protein, the sequence of which consists of 346 amino acids, is 48 kDa. The absence of an optimal conformation in such a large protein can cause steric difficulties in its interaction with the sorbent. Therefore, it is necessary to create a new recombinant polypeptide that binds HSA with the same high activity as A2 polypeptide, but having a smaller sequence and, consequently, lower molecular weight.

According to the claimed invention, a recombinant A4 polypeptide and its producer strain are obtained. Recombinant A4 polypeptide contains a sequence of two HSA-binding GA modules and the W region and has a high ability to bind HSA and lower molecular weight. Its use is possible when creating a sorbent for the effective removal of HSA from blood serum.

The problem was solved by constructing unique primers directed to regions of a gene encoding a polypeptide consisting of two HSA-binding GA modules and the W region. In order to quickly and easily produce recombinant A4 polypeptide, the pQE expression plasmid system was used to clone the DNA fragment. To create a producer strain of the recombinant A4 polypeptide, E. coli M15 strain was used. As a result of experimental work, this strain acquired new properties: the ability to express recombinant A4 polypeptide. The new strain is called E. coli M15-A4. The strain is deposited in the collection of NIIEM SZO RAMS, No. 715.

In the process, a unique recombinant pA4 DNA was obtained by PCR using the chromosomal DNA of group D streptococcus strain DG13, unique primers PA1 and PA2 and subsequent cloning using the expression plasmid pQE-30 (The QIAexpress System, Qiagen, USA).

A recombinant plasmid DNA pQE 30-pA4 carrying the recombinant pA4 DNA and an E. coli M15-A4 producer strain allowing the expression of the recombinant A4 polypeptide under certain conditions were also created.

In the process, a gene fragment was obtained that encodes a region with two HSA-binding GA modules and a region W of strain DG13 of group G streptococcus, designated as pA4, 612 bp in size. by PCR using primers PA1 and PA2 and the chromosomal DNA of strain DG13 of group G streptococcus G. The authors also cloned pA4 using the expression plasmid pQE-30 and subsequent transformation of the resulting recombinant plasmid (designated as pQE 30-pA4) into the heterologous system of E. coli M15. The authors obtained a producer strain of recombinant A4 polypeptide, designated as E. coli M15-A4. The authors also expressed the expression of the recombinant A4 polypeptide by cells of the E. coli strain M15-A4 followed by one-step affinity purification using Ni-Sepharose (GE Healthcare, Sweden).

Obtaining recombinant A4 polypeptide.

The source of chromosomal DNA was the group D streptococcus DG 13 strain G (isolated from cow's milk). DNA isolated by phenol-chloroform extraction was used as a template in PCR to obtain the pA4 gene fragment (612 bp). Oligonucleotide primers are presented in Table 1.

The underlined units of the nucleotide sequence indicate restriction sites.

Analysis of the PCR results was carried out by separation of DNA fragments in a 1% agarose gel using horizontal electrophoresis (figure 1).

The desired amplified DNA region was isolated using the QIAquick Gel Extraction Kit (Qiagen, USA).

The resulting fragment was cloned using the expression plasmid pQE 30. In preparation for cloning, the pA4 fragment (612 bp) and the pQE 30 plasmid isolated from agarose were restricted with BamHI and Kpnl enzymes to form sticky ends. Restriction products were separated by horizontal electrophoresis on a 1% agarose gel, and then ligated.

Calcium transformation of E. coli M 15 strain was carried out by ligation products. The transformation process was carried out according to the E. coli transformation technique using dense selective media containing 100 μg / ml ampicillin, 25 μg / ml kanamycin [Maniatis T, Fritsch EF, Sambrook J. Molecular cloning : a laboratory manual. - Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 867, 1982].

Antibiotic-resistant transformants were split into two parallel Petri dishes with antibiotics. Colonies from one cup were transferred onto a nitrocellulose membrane and opened with a solution containing 0.2 N NaOH, 0.1% SDS and 0.5% β-mercaptoethanol. The membrane was incubated for 1 hour at room temperature in a blocking solution (2 parts of 3% milk and 1 part of phosphate-buffered saline (PBS)) to remove non-specific binding and then in a blocking solution containing HSA labeled with horseradish peroxidase (Sigma, USA).

Conjugation of horseradish peroxidase with HSA was carried out by the periodate method [Fremel G. Immunological methods. M .: Medicine: 438-439, 1987]. Then the membrane was washed sequentially with a blocking solution and PBS. Peroxidase activity was shown with a solution of paraphenylenediamine at a concentration of 1 mg / ml with 0.15% hydrogen peroxide. Recombinant plasmids were isolated from transformants with the most pronounced expression of HSA-binding activity using Mini-prep, plasmid DNA purification kit (Qiagen, USA). The presence of recombinant DNA in them was confirmed in PCR with the initial primers PA1 and PA2. Plasmid DNA pQE 30-pA4 was used as a template in PCR with primers pQEl and pQE2. The amplificate was isolated after electrophoresis on a 1% agarose gel and sequenced.

The final data coincided with the known nucleotide sequence encoding a region with two HSA-binding GA modules [Sjobring U. Infect, and Immun. 60: 3601-3608, 1992].

In FIG. 2a shows the nucleotide sequence consisting of 612 bp pA4 recombinant DNA and 57 bp plasmids pQE 30, and in FIG. 2b is an amino acid sequence consisting of 223 amino acid residues in which 204 amino acids of A4 polypeptide are encoded by recombinant pA4 DNA and the first 19 amino acids are encoded by pQE 30 plasmid. Sequences of HSA-binding GA modules are marked in bold.

A culture of E. coli M 15 strain containing the recombinant plasmid pQE 30-pA4 designated as E. coli M 15-A4 was cultured in BHI liquid medium (Brain Heart Infusion Broth, Gibco, CIIIA) supplemented with antibiotics (ampicillin (100 μg / ml) and kanamycin (25 μg / ml)) until the late logarithmic phase of growth (OD ₆₀₀ = 0.7-0.9). Then, expression of the recombinant protein was induced by the addition of isopropyl-beta-D-thiogalactopyranoside (IPTG), and the cells were cultured for another 4 hours. After this, the cells were precipitated by centrifugation, washed with lysis buffer A (20 mM Na ₂ HPO ₄ , 20 mM NaH ₂ PO ₄ , 500 mM NaCl, 20 mM imidazole, pH 8.0) and suspended in the same buffer, adding a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), to a concentration of 1 mm. The cell suspension was opened with ultrasound and centrifuged. The supernatant was applied to a column filled with Ni-Sepharose. After the protein was bound to Ni-Sepharose, the column was washed with buffer A to remove unbound proteins. Recombinant protein was suirable buffer B (20 mm Na ₂ HPO ₄ , 20 mm NaH ₂ PO ₄ , 500 mm NaCl, 250 mm imidazole, pH 8.0). After affinity chromatography, the protein was dialyzed for 18 hours against distilled water.

The isolated recombinant polypeptide was analyzed by polyacrylamide gel electrophoresis, which made it possible to conclude that the purification of the polypeptide was satisfactory and also its molecular weight by comparing the range of the A4 polypeptide with the range of proteins of known molecular weight (Precision Plus Protein standards (161-0373), Bio-Rad , USA). The molecular weight of A4 polypeptide was equal to (27.0 ± 0.5) kDa (figure 3).

Thus, the recombinant A4 polypeptide contains the amino acid sequence of the HSA-binding group G Streptococcus polypeptide of strain DG 13 containing 204 amino acids covalently linked to 19 amino acid residues encoded by pQE 30.

Study of the interaction of recombinant A4 polypeptide with HSA when compared with a similar reaction with A2 polypeptide

The ability of A4 and A2 polypeptides to bind HSA represented by the following samples: 1) HSA - human polyclonal serum albumin labeled with horseradish peroxidase, 2) unlabeled polyclonal HSA, was evaluated using the direct method of receptor-enzyme analysis (XPA) [Gupalova et al. Biotechnology, No. 3: 75-84, 2012]. Polypeptides were adsorbed onto the plate at a concentration of 1 μg / ml. At the same time, the amount of tagged HSA associated with them was estimated. Figure 4 shows a histogram showing a comparison of HSA-binding activity of A4 and A2 polypeptides. The tested A4 and A2 polypeptides have HSA-binding activity against labeled HSA, and the amount of HSA bound by A4 polypeptide is approximately the same as in the case of A2 polypeptide.

A method for removing HSA and IgG from blood serum, i.e. the creation of affinity carriers: Sepharose 4B-HSA-binding polypeptide and Sepharose 4B-IgG-binding polypeptide.

The preparation of a recombinant polypeptide used to create one of the affinity carriers, an IgG-binding polypeptide, is described in the article [Gupalova et al. Vestnik Akademii Nauk Medical Sciences, 3: 44-49, 1996] and in Patent No. 2056859 “Recombinant IgG-binding G- protein of streptococcus group G "[Gupalova TV, 1996].

As a matrix of affinity carriers, cyanogen bromide 4B sepharose (Sigma) was used. The ligands immobilized on the matrix were recombinant IgG and HSA binding polypeptides.

Previously, this approach has not been used to remove CSA and IgG from serum.

The pre-cyanogen brominated Sepharose 4B was washed several times with cold 1 mM HCl, each time centrifuged, the supernatant was discarded, adding a fresh portion of cold 1 mM HCl. Then, the Sepharose suspension was washed with 0.1 M bicarbonate buffer, pH 8.3.

Then, HSA-binding A2 polypeptides were added to three portions of the prepared, as described above, cyanogen brominated Sepharose 4B [Gupalova TV, Biotechnology, No. 3: 75-84, 2012], A4 and IgG-binding polypeptide, respectively, and incubated at 4 ° C with stirring. After incubation, three columns were filled with Sepharose 4B-HSA-binding polypeptide A2, Sepharose 4B-HSA-binding polypeptide A4 and Sepharose 4B-IgG-binding polypeptide. The columns were washed first with bicarbonate buffer with 0.5% NaCl, pH 8.0, then 0.1 M glycine buffer, pH 2.2 and again with bicarbonate buffer with 0.5% NaCl. Columns were equilibrated with a solution of PBS, pH 7.4 and stored at 4 ° C.

In the preparation of affinity sorbents, it was found that when Sepharose 4 B was immobilized, the HSA-binding A4 polypeptide bound it more due to its lower molecular weight. Therefore, with almost the same HSA-binding activity in A2 and A4 polypeptides (Fig. 3), it is preferable to use an affinity sorbent based on A4 polypeptide.

To check the capacity of the sorbents on two prepared columns with Sepharose 4B-HSA-binding polypeptide A4 and Sepharose 4B-IgG-binding polypeptide, balanced FSB, pH 7.4, 100, 200, 400 and 500 μl of serum were sequentially applied. The completeness of serum release from HSA and IgG was checked by blotting. For this purpose, samples of the initial serum, serum samples not bound to the sorbent and eluted from the sample columns were applied to nitrocellulose membranes (SCs). Some SC membranes were transferred to a blocking solution containing HSA labeled with peroxidase, and others containing IgG labeled with peroxidase, and kept for 40 minutes. Membranes were sequentially washed with blocking solution and PBS. The washed membranes were dried in air and placed in a solution of 3.3 /, 5.5 / -Tetramethylbenzidine (TMB, liquid substrate for membranes, Sigma). Staining occurred within a few seconds.

In serum samples that did not bind to sorbents and passed through affinity columns upon application of 100, 200, 400, and even 500 μl of serum, both HSA and IgG were almost completely absent.

Two affinity columns with 4B-IgG-binding Sepharose and 4B-HSA-binding A4 Sepharose polypeptides were linked. The first of them was applied 400 μl of serum. A sample that did not contact the first column flowed to the second column. Affinity chromatography was performed as described above. Samples of the initial serum, serum that did not bind to the sorbents of both columns, and samples of eluted HSA and IgG were evaluated by blotting. The results of blotting showed, as in the first experiment, the absence of serum in the sample that did not bind to the sorbents, both HSA and IgG (figa and 5b). Removal of HSA and IgG from serum was also verified by SDS-PAGE electrophoresis (Fig.6). As follows from Fig.6, a serum sample after passing it sequentially through two affinity columns, was released almost completely from HSA and IgG (Fig.6, (3)). An IgG sample purified by affinity chromatography eluted from the first column is shown in FIG. 6, (4), a purified HSA sample is shown in FIG. 6, (5).

The sorbent with the attached IgG-binding polypeptide and the sorbent with the attached HSA-binding A4 polypeptide were mixed and a combined sorbent was obtained. On a column with a combined sorbent was applied 400 μl of serum. Affinity chromatography was performed as previously described. Samples of the original serum, serum that did not bind to the sorbent of the combined column and the eluted sample containing HSA and IgG were analyzed by SDS-PAGE electrophoresis (Fig. 7). Serum samples before and after passing it through a column with a combined sorbent were analyzed by 2-DE method (Fig. 8). As can be seen from Fig.7, a serum sample after passing it through an affinity column was freed from HSA and IgG (Fig.7, (4)). 7, (3) shows the presence of HSA and IgG in a serum sample eluted from the column. This sample was then passed through an IgG-binding polypeptide column to separate HSA and IgG from each other and to obtain them in a purified state. The IgG sample was eluted from the column, and the HSA sample passed through the column. As shown in FIG. 7, HSA and IgG were obtained in a highly purified state (FIG. 7, (1 and 2)). Samples of the original serum and serum passed through the column and not bound to the sorbent were analyzed by the 2-DE method, which showed the release of serum from HSA and IgG (Fig. 8 B). The release of serum from HSA IgG allows the detection of proteins located both in the high molecular weight region (70 kDa) shielded by HSA and in the region of 30-50 kDa, where a wide IgG band hides proteins migrating in this region.

Thus, the created sorbents: sepharose 4B-IgG-binding polypeptide, sepharose 4B-HSA-binding A4 polypeptide and a combined sorbent consisting of both sorbents, allow almost completely removing CSA and IgG from serum and detect proteins in serum at low concentrations , and thereby go on to the search and characterization of new biomarkers.

The following are specific examples illustrating some embodiments of the invention, but not limiting it.

Example 1. Obtaining a pA4 DNA fragment by PCR.

To 0.25 μg of genomic DNA isolated by phenol-chromosome extraction from strain DG 13 of group G streptococcus, 10 micromoles of each of the specific primers flanking the test sequence, a buffer with magnesium for polymerase, 0.2 mM of four deoxyribonucleotide triphosphates were added, the volume was adjusted water to 25 μl and 0.5 μl of thermostable DNA polymerase was added. 40 μl of mineral oil was layered on the surface of the liquid. The tubes were placed in an amplifier and incubated at 94 ° C for 2 min. The PCR program consisted of: denaturation at 94 ° C - 30 sec, annealing of the primers - 60 ° C - 1 min and synthesis - 72 ° C - 1 min. This cycle was repeated 30 times, after which the mixture was incubated at 72 ° C for 10 minutes. In the work used oligonucleotide primers are shown in Table 1. PCR products were separated on a 1% agarose gel in horizontal electrophoresis (figure 1). Amplified DNA was isolated using the QIAquick Gel Extraction Kit (Qiagen, USA). The analysis of the size of the obtained DNA fragment was carried out based on a comparison of its electrophoretic mobility with the electrophoretic mobility of the molecular weight marker (100 bp DNA marker, Helikon).

Figure 1 shows the electrophoregram of the amplified pA4 DNA fragment, where 1-100 bp DNA is a marker, (from top to bottom: 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 nucleotide pairs), 2 - PCR product.

As a result of PCR with primers PA1 and PA2, a 612 bp fragment was obtained, which is shown in Fig. 1.

Example 2. Cloning of the pA4 DNA fragment using the expression plasmid pQE 30.

Plasmid pQE 30 and the fragment obtained by PCR were treated with two restriction enzymes BamHI and KpnI, which led to the formation of sticky ends. Restriction products were separated by horizontal electrophoresis on a 1% agarose gel, and then isolated from agarose using the QIAquick Gel Extraction Kit (Qiagen, USA). During cloning, to 5 μl of the pA4 DNA fragment, 1 μl of the pQE 30 DNA fragment obtained after restriction and excised from agarose, 2 μl of ten-fold ligase buffer and 1 μl of T4 phage ligase were added.

The volume was adjusted with distilled water to 20 μl. The mixture was incubated at 10 ° C for 12 hours. As a result of the cloning, a recombinant plasmid designated as pQE 30-pA4 carrying the recombinant DNA (designated as pA4) consisting of 612 bp was obtained. and 57 bp a fragment of plasmid pQE-30.

Example 3. Transformation of the culture of E. coli M 15 plasmid DNA pQE 30-pA4.

The recombinant plasmid was transformed into the heterologous system of E. coli M 15. As a positive control, E. coli M 15 was transformed in parallel with the original plasmid DNA pQE 30.

The genetic marker of plasmid DNA pQE 30 is an amp marker encoding beta-lactase, which provides ampicillin resistance to cells carrying this plasmid. A cell culture of E. coli M 15 was cultured in BHI broth (Brain Heart Infusion Broth, Gibco, USA) with kanamycin (25 μg / ml) for 12 hours at 37 ° C with vigorous stirring. Then the inoculum was subcultured on a new volume of the same medium (10 ml of medium — 0.1 ml of inoculum) and incubated at 37 ° C for 2-3 hours with stirring to OD ₆₀₀ = 0.3. The grown cells in a volume of 1.5 ml were centrifuged for 1 min at 12000 rpm and the resulting pellet was suspended in 200 μl of a solution of 0.1 M CaCl ₂ . Next, the mixture was kept in ice for 1 hour. After centrifugation, the pellet was resuspended in 187.5 μl of 0.1 M CaCl ₂ and 64.5 μl of distilled water. Then 0.2 μg of plasmid DNA was added to the tube and the mixture was incubated in ice for 1 hour. Then the mixture was kept for 2 min at 42 ° C and the mixture was again transferred to ice for 10 min. After that, 1 ml of BHI broth was added to the mixture and incubated for 1 hour at 37 ° C. After precipitation by centrifugation (8000 rpm for 1 min), the cells were plated on plates with 1% L-agar (Difco, USA) containing ampicillin (100 μg / ml) and kanamycin (25 μg / ml). After 18 hours of cell growth at 37 ° C., clones of transformants were selected. Antibiotic-resistant transformants were transferred to two parallel Petri dishes with antibiotics. Colonies from one cup were transferred onto a nitrocellulose membrane and opened with a solution containing 0.2 N NaOH, 0.1% SDS and 0.5% β-mercaptoethanol. The membrane was incubated for 1 hour in a blocking solution containing HSA labeled with horseradish peroxidase (Sigma, USA). Then it was sequentially washed with a blocking solution and a solution of FSB. Peroxidase activity was exhibited by a solution of paraphenylenediamine at a concentration of 1 μg / ml with 0.15 M hydrogen peroxide.

Thus, the data obtained allow us to conclude that, as a result of the cloning, recombinant clones carrying the pQE 30-pA4 plasmids with recombinant pA4 DNA were obtained.

Example 4. Purification of recombinant A4 polypeptide. The culture of E. coli strain M 15-A4 was grown in BHI broth with the addition of ampicillin at a concentration of 100 μg / ml and kanamycin at a concentration of 25 μg / ml overnight with vigorous stirring. Then the cells were subcultured in 800 ml of the same medium and incubated at 37 ° C for 2-3 hours with vigorous stirring to OD ₆₀₀ = 0.7-0.9. The expression of the recombinant protein was induced by the addition of a solution of isopropyl-beta-D-thiogalactopyranoside to a final concentration of 2 mM, after which the cells were incubated under the same conditions for another 4 hours. The resulting cell suspension was centrifuged at 4000 rpm for 20 minutes. The supernatant was decanted and the cells suspended in buffer A (20 mM Na ₂ HPO ₄ , 20 mM NaH ₂ PO ₄ , 500 mM NaCl, 20 mM imidazole, pH 8.0), adding a phenylmethyl sulfonyl fluoride protease inhibitor (PMSF) to a final concentration of 1 mm. For cell lysis, a triple ultrasonic treatment was used at 4 ° C for 20 seconds with a break of 40 seconds in an ultrasonic disintegrator (UZDN-1 U4.2, England). The cell lysate was centrifuged at 20,000 rpm and 4 ° C for 20 minutes. The supernatant was passed through a 0.45 μm filter (Millipore, USA) and then it was applied to a Ni-Sepharose column previously equilibrated with buffer A. The column was then washed with the same buffer until the OD _{280 of the} outgoing solution was greater. than 0.01. The protein was eluted with buffer B solution (20 mM Na ₂ HPO ₄ , 20 mM NaH ₂ PO ₄ , 500 mM NaCl, 250 mM imidazole, pH 8.0). The eluate was collected in fractions of 500 μl and the OD ₂₈₀ values were measured in them. The fractions with the highest OD ₂₈₀ values were pooled and dialyzed against distilled water for 12 hours. Figure 3 presents the electrophoregram of the recombinant A4 polypeptide.

Figure 3 shows the electrophoregram of the recombinant A4 polypeptide, where 1 is the molecular weight marker (from top to bottom: 113, 92, 52, 35, 28 and 21 kDa), 2 is the preparation of purified A4 protein.

The molecular weight of A4 polypeptide was determined by comparing the range of A4 protein with the range of proteins of known molecular weight (Precision Plus Protein standards (161-0373), Bio-Rad, USA). The molecular weight of protein A4 was equal to (27 ± 0.5) kDa.

Example 5. The binding of serum labeled HSA adsorbed polypeptides A2 and A4.

Analysis of HSA-binding activity was carried out by direct XRD. For X-ray diffraction, NUNC MaxiSorp (Danmark) tablets were used.

The preparation of diluted preparations of labeled HSA was carried out using a solution of FSB. Unbound reagents were removed by washing the plates three times with FSB solution with 0.05% Tween 20 (FSBT).

Polypeptides A2 and A4, diluted in PBS, pH 8.0 to 1 μg / ml, were adsorbed onto the plate for 18 hours at 4 ° C. After washing three times, diluted labeled HSA was added to the plates. Incubation was carried out at 37 ° C for 1 hour. Next, the tablets were washed 3 times from unbound reagents. The activity of the horseradish peroxidase label enzyme was determined using the chromogen tetramethylbenzidine (TMB) dissolved in 0.1 M citrate-phosphate buffer, pH 5.0 with the addition of hydrogen peroxide. The reaction was stopped by the addition of 2N sulfuric acid. The optical density was determined at a wavelength of 450 nm on a multiscan.

It was found that the A4 polypeptide, like the A2 polypeptide, has approximately the same high HSA-binding activity, as shown in Fig.4.

Figure 4 presents a comparison of the binding efficiency of A2 and A4 polypeptides with HSA, where the black column is the A2 polypeptide, the white column is the A4 polypeptide, the abscissa axis is the dilution of the labeled HSA, and the ordinate axis is the optical density (OD45o).

Example 6. A method for removing HSA and IgG from blood serum on affinity carriers: Sepharose 4B-IgG binding polypeptide and Sepharose 4B-HSA binding polypeptide.

Two affinity columns with Sepharose 4B-IgG-binding and Sepharose 4B-HSA-binding A4 polypeptides were connected to each other and 400 μl of serum was applied to the first of them. A sample that did not contact the first column flowed to the second column. Affinity chromatography was performed as described above. Samples of the initial serum, serum that did not bind to the sorbents of both columns, and samples of eluted HSA and IgG were evaluated by blotting. The results of blotting showed, as in the first experiment, the absence of serum in the sample that did not bind to the sorbents, both HSA and IgG (figa and 5b).

Figure 5 shows two SCs, one of which is incubated with a HSA-binding polypeptide labeled with peroxidase (Fig. 5a), and the second with an IgG-binding polypeptide labeled with peroxidase (Fig. 5b).

On figa (nitrocellulose membrane with developed spots):

1 - spots corresponding to two-fold dilutions of the original serum,

2 - spots corresponding to dilutions of eluted HSA,

3 - absence of spots corresponding to dilutions of serum that flowed through the affinity column with a sorbent and freed from HSA.

On figb (nitrocellulose membrane with developed spots):

1 - spots corresponding to two-fold dilutions of the original serum,

2 - spots corresponding to dilutions of eluted IgG,

3 - the absence of spots corresponding to dilutions of serum that flowed through the affinity column with a sorbent and freed from IgG.

Removal of HSA and IgG from serum was also verified by SDS-PAGE electrophoresis (Fig.6).

The figure 6 shows the electrophoregram:

1 - molecular weight marker (top to bottom: 113, 92, 52, 35, 28 kDa),

2 - sample of the original serum,

3 - a sample of serum not associated with sorbents on the columns and freed from HSA and IgG,

4 - sample IgG, eluted and purified by affinity chromatography,

5 - a sample of HSA, eluted and purified by affinity chromatography.

Example 7. A method of removing HSA and IgG from blood serum on a combined affinity carrier.

The sorbent with the attached IgG-binding polypeptide and the sorbent with the attached HSA-binding A4 polypeptide were mixed and a combined sorbent was obtained on which both IgG and HSA can be removed from serum. On a column with a combined sorbent was applied 400 μl of serum. Affinity chromatography was performed as previously described. Samples of the original serum, serum that did not bind to the sorbent of the combined column and the eluted sample containing HSA and IgG were analyzed by SDS-PAGE electrophoresis (Fig. 7). Serum samples before and after passing it through a column with a combined sorbent were analyzed by 2-DE method (Fig. 8).

Figure 7 presents the electrophoregram of a sample of the original serum, a sample of a serum that did not bind to the combined sorbent and was freed from HSA and IgG, a sample containing HSA and IgG, eluted from the combined sorbent and samples of HSA and IgG eluted from sorbents containing HSA- binding polypeptide or IgG binding polypeptide. A sample eluted from the combination column containing HSA and IgG was passed through the IgG binding polypeptide column to separate the HSA and IgG from each other and to obtain them in a purified state. The IgG sample was eluted from the column, and the HSA sample passed through the column.

In Fig.7 SDS-PAGE electrophoresis:

1 - sample IgG, eluted and purified by affinity chromatography;

2 - a sample of HSA, eluted and purified by affinity chromatography;

3 - a sample eluted from a combined sorbent containing HSA and IgG;

4 - a sample of serum that is not associated with sorbents on the columns and freed from HSA and IgG;

5 - sample of the original serum;

6 - molecular weight marker (top to bottom: 113, 92, 52, 35, 28 and 21 kDa). On Fig shows electrophoregrams after conducting 2DE. On figa-2DE source serum ellipses are proteins:

1 - HSA;

2 - IgG, heavy chains;

3 - IgG, light chains.

On figb-2DE samples of serum freed from HSA and IgG. The release of serum from HSA and IgG allows the detection of proteins located both in the high molecular weight region (70 kDa) shielded by HSA and in the region of 30-50 kDa, where a wide IgG band hides proteins migrating in this region.

Claims (8)

1. Recombinant pA4 DNA having the nucleotide sequence shown in FIG. 2a encoding a recombinant A4 polypeptide having the ability to selectively bind human serum albumin - HSA. 2. Recombinant plasmid DNA pQE 30-pA4, which is a plasmid pQE 30, carrying the recombinant pA4 DNA according to claim 1, and providing a recombinant A4 polypeptide. 3. The strain Esherichia coli M 15-A4, producing a recombinant A4 polypeptide, obtained on the basis of the strain Esherichia coli M15 and containing the recombinant plasmid DNA pQE 30-pA4 according to claim 2. 4. Recombinant A4 polypeptide having the ability to selectively bind HSA and characterized by the amino acid sequence shown in FIG. 26, in which the first 19 amino acids are encoded by the sequence of plasmid pQE 30, covalently linked to the next 204 amino acids encoded by the sequence of the HSA-binding fragment of the chromosomal DNA of strain DG 13 streptococcus group G-CTG. 5. An affinity sorbent consisting of a mixture of a matrix consisting of 4 V cyanbrom-activated sepharose pre-washed with cold ImM HCl and 0.1 M bicarbonate buffer, pH 8.3, and a solution of the ligand — HSA-binding A4 polypeptide A4 added to it, according to .4, which is incubated with constant stirring for 18 hours at 4 ° C. 6. The affinity combined sorbent obtained by mixing equal amounts of two sorbents: the affinity sorbent according to claim 5 and a similar affinity sorbent, in which the known recombinant IgG-binding polypeptide is used as a ligand. 7. A method of sequential removal of HSA and IgG from blood serum by affinity chromatography, characterized in that the serum sample is passed through a first column containing an affinity sorbent, in which a recombinant IgG-binding polypeptide is used as a ligand, and then passed through a second one connected to it a column containing an affinity sorbent according to claim 5. 8. A method for the simultaneous removal of HSA and IgG from blood serum, characterized in that the serum sample is passed through a column containing the affinity combined sorbent according to claim 6.

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