RU2448116C2 - Method for producing active fragment of human alpha-fetoprotein - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is offered is a method for recovery and purification of a fragment of human alpha-fetoprotein. The method involves recovery of inclusion bodies containing a target protein from a producer strain biomass, solubilisation followed by renaturation and purification of the active fragment of human alpha-fetoprotein.

EFFECT: invention is applicable for producing the active fragment of human alpha-fetoprotein for the purpose of making anticancer preparations of directed action for selective tumour cell destruction.

5 dwg

Description

The invention relates to medicine and can be used to isolate and purify the active fragment of human alpha-fetoprotein, expressed as recombinant inclusion bodies, from the biomass of the producer strain, which can be used in the preparation of targeted antitumor drugs for the selective destruction of tumor cells.

A known method, including processing the initial tissue with butyl alcohol, centrifugation at 6000 rpm for 30 minutes, dialysis of the aqueous protein phase against a 0.9% sodium chloride solution, affinity chromatography of the dialyzed aqueous protein phase on immobilized estrogens, followed by elution with butyl-alpha-fetoprotein alcohol or a mixture of butyl alcohol with a buffer solution, while the yield of the target product is 60-70%, and the purity is 90-95% [SU 583536, A61K 37/02, 05/10/1979].

The disadvantage of this method is the insufficient yield and purity of the target product.

There is also a known method, including grinding and homogenizing embryonic tissue of human embryos of 8-12 weeks, obtained by medical abortion, centrifuging the homogenate at 5000 rpm, collecting the supernatant, clarifying the supernatant by centrifugation at 12000 rpm, purification of the target product by chromatography on DE-cellulose purification of the target product by chromatography on Sephadex G-100 with cybacron blue dye F36A immobilized on it; purification of the target product by rechromatography on Sephadex G-100 with dye immobilized on it m Cybacron blue F36A and post-treatment of the target product by chromatography on DE-cellulose [K.Hause et al. Clinica Chemica Acta, 133, 1983, 335-340].

The disadvantages of this technical solution are the length and complexity of the process of isolating alpha-fetoprotein, significant changes in the composition of the pool of alpha-fetoprotein and the denaturation of some molecules, leading to a low yield of the final product (25-30%) and to a decrease in its quality (drug purity 85-90% ), as well as low storage stability, as well as possible contamination with viruses, for example, human immunodeficiency virus.

The closest in technical essence to the proposed one is a method that includes clarification of the feedstock by centrifugation and chromatographic purification of the target product on an affinity sorbent, moreover, before centrifugation, sodium chloride is added to the feedstock to a final concentration of 0.5 M, and chromatographic purification is carried out sequentially in three stages, first on an immunoaffinity sorbent containing polyclonal antibodies to alpha-fetoprotein, while elution of alpha-fetoprotein from the sorbent is carried out with a 4 M chloride solution magnesium, the obtained alpha-fetoprotein fraction is additionally purified by ultrafiltration and gel filtration, and then the eluate is chromatographed on an immunoaffinity sorbent containing a complex of polyclonal antibodies against human blood serum proteins and animal serum immunoglobulins, then sodium chloride is introduced into the target product and the polysaccharide is sterilized by filtration and freeze-dried or stored as a solution.

In addition, for the preparation of the affinity sorbent, BrCN-Sepharose or BrCN-Agarose is used, the immuno-affinity resin in the first chromatography stage contains 5-10 mg / ml of polyclonal antibodies to alpha-fetoprotein, depleted against human serum proteins, and in the second immuno-affinity chromatography stage 5-10 mg / ml of a complex of antibodies against human serum proteins and against animal serum immunoglobulins used to produce antibodies to alpha-fetoprotein, a polysaccharide is used as a polysaccharide type dextran, selected from the group: polyglucin, reopoliglukin, reomacrodex, freeze drying of the target product is carried out in the presence of 5-50 mg / ml sodium chloride and 2-30 mg / ml polysaccharide, and sodium chloride is introduced into the liquid form of alpha-fetoprotein in a concentration 9-50 mg / ml and a polysaccharide at a concentration of 10-50 mg / ml [RU 2123009, C1, A61K 35/14, 12/10/1998].

The disadvantage of this method is the relatively low frequency of the target product, due to the high possibility of contamination with viruses, for example, human immunodeficiency viruses.

The desired result is to reduce the possibility of virus contamination.

The desired result is achieved by the fact that in the method of isolation and purification of the active fragment of human alpha-fetoprotein expressed in the form of recombinant inclusion bodies from the biomass of the producer strain, including the isolation of inclusion bodies containing the target protein, the inclusion bodies containing the target protein are solubilized with subsequent renaturation and purification of the active fragment of human alpha-fetoprotein.

In addition, the required technical result is achieved in that the isolation of inclusion bodies containing the target protein from the biomass of the producer strain is carried out by resuspension in phosphate-buffered saline (PBS), pH 7.4, NaCl 0.1 M, AEBSF (4- (2-Aminoethyl ) benzenesulfonyl fluoride hydrochloride) 1 mM, the resulting suspension was subjected to double ultrasonic treatment for 2 min in a pulse of 0.3 sec at 0 °, followed by addition to the Triton X-100 suspension to a concentration of 1% and the ultrasonic treatment was carried out in the same mode, after which the suspension is centrifuged at 10,000 g for 10 min and the resulting precipitate is suspended in phosphate-buffered saline (PBS), pH 7.4, NaCl 0.1 M, Na-deoxycholate 0.1%, after which the suspension is subjected to ultrasonic treatment in the same mode, centrifuged again at 10,000 g for 10 min, the precipitate is resuspended in phosphate-buffered saline, pH 7.4 and finally centrifuged at 10,000 g for 10 min.

In addition, the required technical result is achieved by the fact that the solubilization of inclusion bodies containing the target protein is carried out by suspension in a denaturing buffer of 0.05 M Na ₂ BO ₃ (pH 8.6), 8 M urea, 0.1 M NaCl, 50 mm B-mercaptoethanol, incubated the resulting solution for 2 hours at room temperature with constant stirring until the precipitate is completely dissolved, after which it is centrifuged for 15 min at 13400 g and 7 ° C.

In addition, the required technical result is achieved in that the renaturation and purification of a fragment of human alpha-fetoprotein is carried out by rapid dilution of the solubilized protein in a renaturation buffer containing phosphate-buffered saline (PBS), pH 8.0, 5 mM, glutathione reduced (Fluka), 1 mM glutathione oxidized (Fluka).

Figure 1 shows the purified recombinant fragment of human AFP, where 1 is the molecular weight standard, 2, 3 is the weight standard (BSA 0.5 and 1.0 μg, respectively), 4, 5, 6, 7 is the purified renatured fragment of human AFP, .2 - analysis of an AFP fragment by reverse phase chromatography, where the ordinate is absorbance at 214 nm, the abscissa is the elution time with an acetonitrile gradient (Waters Breeze HPLC), Symmetry300 C4 column 5 μm 3.9 × 150 mm Column, flow rate 1 ml / min, peak 16 min corresponds to the purified renatured fragment of the AFP person, figure 3 - analysis of the aggregate state of the AFP fragment by gel filtration, where the ordinate is absorbance at 214 nm, the abscissa is time (Waters Breeze HPLC, BioSuite 450 HR SEC column 7.8 × 300 mm, balanced by phosphate-buffered buffer, buffer flow rate 1 ml / min, peak 14 min corresponds to the purified renatured fragment of human AFP), Fig. 4 shows the binding of the FITC-labeled fragment of human AFP to human ovarian adenocarcinoma cells SCOV3 and human peripheral blood lymphocytes, Fig. 5 shows the amino acid sequence of the cloned AFP gene construct, where the bold font is the tandem of arginines (RR).

A method for isolation and purification of the active fragment of human alpha-fetoprotein expressed in the form of recombinant inclusion bodies from the biomass of the producer strain is implemented as follows.

First, we give the theoretical justification of the proposed method.

One of the main directions of modern pharmacology is the directed transport of drugs to specific loci of the human body. The advantage of this approach is the ability to reduce the effective dose of the drug, because instead of passive diffusion, the drug is directed to the target cells.

Of fundamental importance for this approach is the creation of chemically or biologically engineered macromolecules consisting of a vector part and a non-specific part, which is a drug. Molecules with a specific affinity for target cells should be used as the vector part. Monoclonal antibodies (as well as polyclonal) specific for one or another antigen on the surface of the target cell can be used as such a vector part (1). For use in oncological diseases, the vector part must specifically bind to components (receptors) on the surface of cancer cells. A prerequisite is the almost complete absence of these components (receptors) on the surface of normal cells. The use of antibodies as a vector part has faced a number of problems. Without dwelling on them, it can be pointed out that, despite many years of work for approximately 30 years, not a single similar drug has passed clinical trials (1). An inevitable disadvantage of immunoconjugates is the possibility of provoking an immune response.

There is another class of biological macromolecules specific for cancer cells, the so-called oncofetal proteins (2). These proteins are not foreign to this organism, and receptors for them are present mainly in cancer cells. This group of proteins includes alpha-fetaprotein (AFP) (3), cancer-embryonic antigen, placental alkaline phosphatase, basic fetaprotein, as well as its alpha, beta and gamma forms, pancreatic fetal protein. A distinct advantage of AFP is that it specifically binds and is endocytosed by tumor cells; at the same time, the number of receptors for it on normal human cells is very low (4-6). Under normal conditions, AFP is synthesized during embryonic development. The main synthesis sites are the fetal liver and the visceral endoderm of the yolk sac. The maximum AFP concentration is reached in human fetal serum (up to 10 mg / ml) (7). The main function of AFP is the transport of fatty acids, heavy metals, bilirubin, and a number of other components (8). In the human blood in the postnatal period, the AFP level does not exceed 10 ng / ml (7). During this period, the main transport function in the blood is performed by serum albumin. An increase in the level of AFP in the blood most often indicates the appearance of cancer cells (liver tumors, teratocarcinomas, intestinal tumors) (4-6, 9).

AFP is a fairly well-studied protein. It consists of one polypeptide chain of molecular weight 74 kDa (10). The protein is glycosylated, carbohydrates make up from 3% to 4.3%. They include glucose, galactose, mannose, n-acetylglucosamine and sialic acid. The microheterogeneity of AFP associated with the composition of the glycosidic part is experimentally shown. Three domains are distinguished in the spatial structure of a protein by homologous computer modeling. The greatest homology of AFP is with serum albumin, the spatial structure of which has been determined and is used to model the spatial structure of AFP.

The accumulation of AFP in cells occurs as a result of receptor-mediated endocytosis of this protein. In the process of endocytosis, AFP is first found in clotrin vesicles, and then in endosomes and folded membranes of the central region of the Golgi apparatus (5). In this case, the bulk of the AFP does not undergo degradation, but again goes into the extracellular environment.

An AFP receptor has been identified. Receptors were found in tumor cells of cancer of the liver, lungs, stomach, breast, etc. (5, 6). By immunological methods, the receptor was found on the surface of the cells of these types of tumors; the content of the receptor correlated with the malignancy of the tumor. In normal tissue cells, the AFP receptor was not detected (4). These circumstances fully meet the requirements for a vector molecule for targeted drug delivery. Indeed, the use of AFP as a vector part for the targeted transport of antitumor antibiotics to cancer cells ensured high efficiency and selectivity. The pronounced positive effect of AFP conjugates with cytostatics such as doxorubicin, esperamycin and vinblastine has been shown in the treatment of animals with experimental AFP tumors (11-13).

There are various methods for combining the vector part and the drug. Non-covalent bonds are sometimes used, but, as a rule, preference is given to the covalent connection of the parts of the resulting conjugate. The covalent bonding of the parts of the conjugate can be achieved using various chemical bonds. On the one hand, the covalent compound must be stable enough to ensure the delivery of the conjugate to the site of action, while taking into account not only the chemical stability of this bond, but also the possibility of its destruction by enzymes. On the other hand, the covalent bond should not interfere with the functioning of the drug, and in the best case should be destroyed when the conjugate enters the cell. Were used methods of conjugation of AFP with drugs, a distinctive feature of one of which is that the amino group of the AFP is modified with succinimidyl trans-4- (N-maleimidylmethyl) cyclohexane-1-carboxylate (SMCC) or N-hydroxysuccinimide ester of 3-maleimidebenzoic acid MBS), after which the obtained derivative is cross-linked with the SH groups that are present initially or are additionally introduced using 3- (2-pyridyldithio) propionic acid succinimide ester (SPDP) (14). Another method for the preparation of the conjugate involves condensation of the amino groups of the AFP with the aldehyde group initially contained in the drug or additionally introduced thereto after periodate activation and subsequent reduction of the intermediate with borohydride.

The AFP conjugates obtained with these methods with several different cytostatics, for example, doxorubicin and calichemicin, had a selective effect on target cancer cells (14).

The use of natural human AFP is associated with a number of technical, medical and ethical limitations. Natural AFP is released from abortive material. This method of isolation is prohibited or restricted in most countries due to possible virus contamination, as well as due to non-compliance with modern ethical requirements. As a result, the use of recombinant AFP is attractive. Moreover, it is possible to use not full-sized AFP, but its fragments, determined based on the results of computer modeling. This follows from the fact that certain fragments of AFP retained the ability of natural AFP to accumulate in tumors (15, 16).

Consider an example of the implementation of the proposed method for the isolation and purification of the active fragment of human alpha-fetoprotein expressed in the form of recombinant inclusion bodies from the biomass of the producer strain.

Isolation and purification of the active fragment of human alpha-fetoprotein from the biomass of the producer strain was carried out taking into account the optimization of the stages of the proposed method in order to increase the yield of the final product.

Stage A. Isolation of inclusion bodies containing the target protein from the biomass of the producer strain.

The biomass of the producer strain was resuspended in a buffer of the following composition: phosphate buffered saline (PBS), pH 7.4, NaCl 0.1 M, AEBSF (4- (2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) 1 mM.

The suspension was subjected to sonication for 2 min (pulse 0.3 sec) at 0 ° C. The procedure was repeated twice. Then, Triton X-100 was added to the suspension to a concentration of 1% and again subjected to sonication in the same mode. Then, the suspension was centrifuged at 10000 g (Beckman centrifuge, JA-14 rotor) for 10 min, the precipitate was suspended in a buffer of the following composition: phosphate buffered saline (PBS), pH 7.4, NaCl 0.1 M, Na-deoxycholate 0.1%. The suspension was sonicated in the same mode, then centrifuged again at 10,000 g for 10 minutes. The pellet was resuspended in phosphate-buffered saline, pH 7.4, and centrifuged again. The resulting inclusion bodies were either used immediately in further steps or frozen at -70 ° C.

The presence of the target protein in inclusion bodies during their purification, as well as possible losses, was monitored by electrophoresis under denaturing Lemmli conditions. At this stage, the purity of the target protein for the protein component was 70-80%.

Stage B. Solubilization of inclusion bodies containing the target protein.

The sediment of inclusion bodies was suspended in denaturing buffer of the following composition: 0.05 M Na ₂ BO ₃ (pH 8.6), 8 M urea, 0.1 M NaCl, 50 mM B-mercaptoethanol. The solution was incubated for 2 hours at room temperature with constant stirring until the precipitate was completely dissolved. Then it was centrifuged for 15 min at 13,400 g and 7 ° C (Beckman centrifuge, JA-14 rotor).

Stage B. Renaturation and purification of a fragment of human AFP.

Renaturation of a fragment of recombinant human alpha-fetoprotein was carried out by rapid dilution of the solubilized protein into a renaturation buffer containing phosphate-buffered saline (PBS), pH 8.0, 5 mM glutathione reduced (Fluka), 1 mM glutathione oxidized (Fluka). The final concentration of the recombinant protein was 0.01-0.02 mg / ml. Renaturation was carried out at + 4 ° C for 2 days. Reverse phase chromatography was used to determine the degree of renaturation of a fragment of human AFP.

At the next stage, the protein was purified by metal chelate chromatography on a Chelating Sepharose FF sorbent (Amersham, UK). The process consisted of preparing a chromatographic resin, applying a protein solution from the renaturation, washing and elution stages. All procedures are performed at 4 ° C.

The sorbent charged with Ni ² + and equilibrated with renaturation buffer was added to the diluted protein solution and incubated for 1 hour. Then, the sorbent with bound protein was transferred to a chromatographic column and washed with a buffer containing phosphate-saline buffer (PBS), pH 8.0, 15 mM imidazole in a volume exceeding the volume of the sorbent by 20-50 times. The quality of washing was checked by spectrophotometric method, measuring the light absorption at a wavelength of 280 nm (protein impurities) and 260 nm (DNA and RNA impurities), taking the above washing buffer as a zero absorption level.

Protein elution from the sorbent was carried out with a buffer containing phosphate-buffered saline (PBS), pH 8.0, 300 mM imidazole.

The resulting solution of the target protein was then dialyzed twice against 500 volumes of phosphate-buffered saline (PBS), pH 8.0. For dialysis, a Spectrum Lab., Inc., US & Canada, MWCO dialysis bag was used: 6-8,000. Each dialysis cycle was carried out at 4 ° C and with constant stirring during the day. Then the protein solution was transferred into a sterile container, frozen and stored at -70 ° C.

The presence of the target protein was monitored after electrophoretic separation of the proteins in a polyacrylamide gel.

Determination of physicochemical and biological properties of a fragment of human AFP.

Determining the concentration of a fragment of human AFP.

The protein concentration in the analytical sample of the preparation after dialysis was determined using a standard set of BCA (Bicinchoninic acid protein assay kit, Sigma).

Determination of the amount of unbound sulfhydryl groups in the molecule of the obtained fragment of human AFP.

The determination of the amount of unbound sulfhydryl groups in the protein molecule was determined using 5.5'-dithio-bis- (2-nitrobenzoic acid) - Ellman's reagent (DTNB) [Ellman G.L. Tissue sulfhydryl groups. // Arch. Biochem. Biophys. - 1959. - No. 82 [1]. - P.70-77.]. As a result of the reaction of Ellman's reagent with sulfhydryl groups, a colored anion is formed, the concentration of which is determined by absorption in the visible spectral region (412 nm).

Analysis of an AFP fragment by reverse phase chromatography.

High-performance reverse phase chromatography was used to determine the degree of renaturation of the human AFP fragment. For this, samples were taken from the protein diluted in the protein renaturation buffer at the initial time, after 1 hour, after a day, and after two days. Waters Breeze HPLC Symmetry300 C4 5 µm 3.9 × 150 mm Column was used for analysis. Before chromatography, the column was balanced with 20 volumes of a solution of 0.1% TFA (trifluoroacetic acid) in water. Then, an aliquot of protein containing 0.1% TFA in water was added, and the column was again balanced with 20 volumes of a solution of 0.1% TFA in water. Protein elution was carried out by a linear gradient of buffers A and B.

A: 0.1% TFA in water. B: 0.1% TFA in acetonitrile.

Buffer flow rate 1 ml / min, total time 100 min. Protein detection was carried out by optical absorption at 280 and 214 nm. It was shown that at the final stage of renaturation, a peak appears with a maximum yield of about 48% acetonitrile, while at the initial stage there are a number of peaks with maximums of yield from 50 to 70% acetonitrile. This peak corresponds to the renatured state of the protein (Figure 2).

Analysis of the state of aggregation of a fragment of human AFP.

The analysis of the state of aggregation of the protein in the preparation was performed using the gel filtration method. Waters BreeSuite 450 HR SEC 7.8 × 300 mm column was used for analysis. The column was balanced with phosphate-buffered saline (10 volumes), a sample (30 μg) was added in a volume of 40 μl, and the flow rate of the buffer was 1 ml / min. Protein was detected by absorption at 280 nm and 214 nm. Proteins and compounds of known molecular masses were used as standards: ferritin (440 kDa), bovine serum albumin (134 and 67 kDa for dimer and monomer, respectively), CD81 protein (30 kDa), lysozyme (14 kDa), NaN ₃ solution.

Chromatography of a human AFP fragment revealed one major protein peak with mobility between CD81 (30 kDa) and lysozyme (14 kDa) (Figure 4) with a peak center of 14.0 min and an additional peak of 11.0 min corresponding to the dimer of a human AFP fragment. The molecular weight calculated by the yield time of a human AFP fragment using standard calibration yields 25 kDa, which is close to the theoretical molecular weight of this polypeptide. High molecular forms and aggregates were absent in detectable amounts.

Determination of the secondary structure in the preparation of a fragment of a human AFP by circular dichroism.

The secondary structure of the protein was studied using circular dichroism spectroscopy (CD) on a JASCO-600 spectropolarimeter (Japan) in the wavelength ranges from 200 nm to 250 nm. The molar ellipticity values were calculated from the equation:

[θ] = [θ] _rev M _ost / (LC),

in which C is the protein concentration (mg / ml), L is the optical path length of the cuvette (mm), [θ] _ism is the measured ellipticity (degrees), and M _ost is the average molecular weight of the peptide residue (Yes) calculated from its amino acid sequence . The measurements were carried out in a 0.1 mm cuvette at a protein concentration of 0.41 mg / ml.

The results show that the protein has pronounced absorption peaks at 190, 208 and 222 nm. This is consistent with a predominantly alpha-helical structure (Woody RW, 1994. Circular dichroism of peptide and proteins. In: circular dichroism: principles and applications). The signal intensities indicate that the protein is very well structured.

The spatial structure of alpha-fetoprotein is not defined. The works proceed from the fact that the protein has a spatial and secondary structure close to its homologue - serum albumin, the spatial structure of which has been studied. Based on these data, it is believed that alpha-fetoprotein is a protein with a secondary structure mainly from alpha-helices. The literature describes an analysis of the secondary structure of alpha-fetoprotein by the circular dichroism method (S. Leong & A. Middelberg, 2006). Both for the native protein and its recombinant form, after renaturation, similar profiles of circular dichroism with pronounced absorption peaks at 190, 208, and 222 nm were obtained, corresponding, as the authors indicate, mainly to the alpha-helical structure. The general ellipticity profile, the distribution of the main peaks and their intensities presented for the full-sized alpha-fetoprotein in this work correspond to our data for the recombinant third domain of human AFP. Although the data for a full-sized protein and its part, one of the domains, should be compared with caution, we can conclude that the target protein we obtained, a fragment of human AFP, has a structure similar to that in the native protein.

Confirmation of the identity of the obtained fragment of human AFP by mass spectrometry.

Confirmation of the identity of the obtained preparation of a fragment of human AFP was performed by mass spectrometric analysis. The essence of the analysis consists in the splitting of the initial protein into fragments under the influence of trypsin, followed by identification of the obtained fragments by the MALDI method, one of the types of mass spectrometric analysis. This approach is one of the main for the identification of proteins.

During the analysis, 9 peptides of alpha-fetoprotein were identified. In accordance with the accepted criteria, the analyzed protein is identified as a fragment of human alpha-fetoprotein.

Analysis of the N-terminal sequence of the preparation of a fragment of human AFP.

A sample, 200 μl of the target protein preparation after chromatography in reverse phases (see above), was concentrated 4 times on a SpeedVac instrument. To determine the N-terminal sequence of the protein, the Prosice 492 protein sequencer (Applied Biosystems) was used with the program and manufacturer's reagents.

The expected sequence of the target protein we obtained, starting with the initiator methionine, in single-letter form is MYIQES. The presence of initiating methionine is necessary for the synthesis of almost any protein on the ribosome. In most proteins, the initiating methionine is partially or completely cleaved, this is also true for proteins expressed in E. coli.

The protein preparation sequence obtained during the analysis is YIQES, i.e. it is as expected without initiating methionine. It should be recalled that a sequence of 7 histidine residues was inserted into the C-terminus of the protein for affinity protein isolation by metal chelate chromatography. The complete amino acid sequence of the cloned construct is shown in FIG.

Analysis of the effectiveness of purification of a fragment of a human AFP from endotoxin.

Our proposed method for isolating a biologically active recombinant fragment of human AFP allows us to effectively separate the protein preparation from endotoxin.

To analyze the content of cell wall lipopolysaccharides (LPS), or endotoxin, in the preparation of a human AFP fragment, a LAL test was performed using the standard protocol attached to the kit for determining LPS. A kit of the company Associates of Cape Cod Incorporated (USA) was used with a declared sensitivity of 0.03 EU / ml, where EE is the unit of endotoxin.

Analysis of the content of cell wall lipopolysaccharides (LPS) in the preparation of a human AFP fragment showed that the amount of endotoxin does not exceed ≈1500 EE per 1 mg of protein product.

Testing the functionality of a fragment of human AFP: specific binding and endocytosis in cells carrying the alpha-fetoprotein receptor.

The accumulation of AFP in cells occurs as a result of receptor-mediated endocytosis of this protein. An AFP receptor has been identified. Receptors were found in tumor cells of cancer of the liver, lung, stomach, breast, etc. (Villacampa MJ et al (1984) Alpha-fetoprotein receptor in a human breast cancer cell line Biochem. Biophys. Res. Commun., 122: 1322- 1327; Moro R. et al (1993) Monoclonal antibodies directed against a widespread oncofetal antigen: The alpha-fetoprotein receptor. Tumor Biol., 14: 116-130). By immune methods, the receptor was found on the surface of the cells of these types of tumors; the content of the receptor correlated with the malignancy of the tumor. In normal tissue cells, the AFP receptor is not detected (Nitsvetov MB et al. (2005) study the expression of AFP receptor in tumor and normal human tissues using the immunohistochemical method. Immunology. 26: 122-125).

The specificity of the binding and penetration into the cells of the obtained recombinant fragment of human AFP was verified using SKOV3 human ovarian adenocarcinoma cells. The control was peripheral blood lymphocytes that did not carry AFP receptors. For analysis, a conjugate of a human AFP fragment with a fluorescent label - fluorescein (FITC) was used. FITC labeled protein was prepared according to standard procedures [Horisberger, M. (1984). In Immunolabeling for Electron Microscopy. Polak, J., Vamdel, I. Ed. Elsevier: Amsterdam, p. 98].

It is generally accepted that at a temperature of + 4 ° C proteins bind to the corresponding receptors on the surface of the cells, if any, but do not penetrate inside. At physiological temperature, proteins bound to receptors undergo endocytosis, i.e. penetrate the cells. The conjugate of the human AFP fragment with FITC (AFP3d-FITC) binds effectively at + 4 ° C to SKOV3 human ovary adenocarcinoma cells that have AFP receptors, but not to lymphocytes that do not have the corresponding receptors. Obviously, this result indicates a specific binding of AFP3d-FITC to a specific receptor for AFP on the surface of SKOV3 cells. At + 37 ° C, a high level of AFP3d-FITC endocytosis in SKOV3 cells is observed, in comparison with this, AFP3d-FITC endocytosis in lymphocytes is at a very low level. The fluorescence level in SKOV3 cells at + 37 ° С is significantly higher, which corresponds to the data on endocytosis of full-sized AFP in these cells. This suggests that with endocytosis, the AFP receptor can recycle, ensuring the penetration of several molecules of the human AFP fragment.

Thus, due to the improvement of the known method for isolation and purification of the active fragment of human alpha-fetoprotein, a higher quality of the target product is obtained, since the possibility of contamination with viruses, for example, human immunodeficiency viruses, is practically excluded.

Literature

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Claims (1)

The method of isolation and purification of the active fragment of human alpha-fetoprotein expressed in the form of recombinant inclusion bodies from the biomass of the producer strain, consisting of the selection of inclusion bodies containing the target protein from the biomass of the producer strain, characterized in that solubilization of inclusion bodies containing the target protein, followed by renaturation and purification of the active fragment of human alpha-fetoprotein, the isolation of inclusion bodies containing the target protein from the biomass of the producer strain is carried out it is carried out by resuspension in phosphate-buffered saline (PBS), pH 7.4, NaCl 0.1 M, AEBSF (4- (2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) 1 mM, the resulting suspension is subjected to two times sonication for 2 min pulse 0.3 s at 0 °, followed by adding to a Triton X-100 suspension to a concentration of 1% and sonication is carried out in the same mode, after which the suspension is centrifuged at 10,000 g for 10 minutes and the resulting precipitate is suspended in phosphate-buffered saline ( PBS), pH 7.4, NaCl 0.1 M, Na-deoxycholate 0.1%, after which the suspension is subjected to sonication in t In the same mode, centrifuged again at 10,000 g for 10 min, resuspended sediment in phosphate-buffered saline, pH 7.4 and finally centrifuged at 10,000 g for 10 min, while solubilization of inclusion bodies containing the target protein is carried out by suspension in denaturing buffer 0.05 M Na ₃ BO ₃ (pH 8.6), 8 M urea, 0.1 M NaCl, 50 mM B-mercaptoethanol, the resulting solution is incubated for 2 hours at room temperature with constant stirring until complete dissolving the precipitate, after which it is centrifuged for 15 min at 13400 g and 7 ° C, while renaturation is carried out by quickly diluting the solubilized protein in a renaturation buffer containing phosphate-buffered saline (PBS), pH 8.0, 5 mM, reduced glutathione (Fluka), 1 mM oxidized glutathione (Fluka).

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