RU2794889C1 - Magnetic affinity sorbent for isolation of recombinant proteins - Google Patents

Abstract

FIELD: sorbents.

SUBSTANCE: present invention relates to a magnetic affinity sorbent for isolation of recombinant proteins, characterized in that it consists of starch-activated magnetic iron oxide nanoparticles with an average size of 11.5 nm, a saturation magnetization at room temperature of 29.8 emu/g, a blocking temperature close the to room temperature, one gram of which allows isolating 100-590 mg of recombinant MBP-containing hybrid proteins from the lysate of E. coli cells.

EFFECT: increased specific sorption capacity of a magnetic affinity sorbent, which is starch-activated magnetic iron oxide nanoparticles.

1 cl, 2 tbl, 5 dwg

Description

The invention relates to the field of biotechnology and is intended for the purification of recombinant proteins of practical interest.

One of the most important areas of modern biotechnology is the production of proteins for biomedical applications using bacterial synthesis. At the same time, the problems of the correct formation of the spatial structure of the target protein, as well as simple and effective purification, are solved. To increase the solubility of target proteins, so-called hybrid proteins are obtained by genetic engineering, in which the gene of the target protein is fused in the same reading frame with the genes of auxiliary polypeptides, which provide higher protein solubility in the bacterial cytoplasm, as well as independent "folding" of the target and auxiliary polypeptides. In addition, auxiliary polypeptides provide rapid isolation of the target protein using chromatography on specially designed affinity sorbents [1]. As auxiliary polypeptides, low molecular weight (for example, polyhistidine, His-tag, 0.84 kDa), medium-sized, for example, streptavidin-binding peptide (SBP-tag, 4.03 kDa) and relatively large polypeptides are used, including single-chain maltose-binding protein (maltose -bindingprotein, MBP, 40 kDa) isolated from E. coli K12. Technologies using MBP-containing hybrid proteins have been used to obtain a wide variety of proteins [2-4]. Appropriate plasmid vectors for the production of hybrid constructs are now commercially available (eg, Gateway Cloning, Invitrogen). MBP is widely used as a fusion partner in the production of a recombinant protein in bacterial cells to increase the expression level, improve the solubility and folding of the target protein [5], and also to provide its one-step purification on amylose-containing carriers. Purification is carried out under physiological conditions, and elution under mild conditions preserves the activity of the target protein. Mild conditions make it possible to purify even intact protein complexes. Due to the high specificity of binding to the MBP sorbent, high purity of the eluted protein is achieved in just one chromatography step [6, 7].

For the purification of MBP-containing hybrid proteins by column chromatography, sorbents are used, which are cross-linked amylose [6], preparations of which are supplied to the market by foreign biotechnological companies (Fisher Scientific, New England BioLabs, etc.).

Magnetic nanoparticles (MNPs) are widely used in various fields of biotechnological and biomedical research [8], including as an affinity sorbent for the isolation of various target molecules, due to such important properties as ease of preparation methods, small size (less than 100 nm) , high specific surface area and, as a result, high sorption capacity, chemical stability. The presence of magnetic properties makes it easy to isolate and concentrate biomolecules of interest using volumetric magnetic separation. This reduces sample preparation time compared to other purification and concentration methods, avoids the need for expensive chromatographic equipment and columns, as well as centrifugation and filtration. Among a wide selection of magnetic nanoparticles (based on metals Co, Fe, Ni, iron oxides (Fe ₃ O ₄ , Fe ₂ O ₃ ), ferrites (MgFe ₂ O ₄ , CoFe ₂ O ₄ , MnFe ₂ O ₄ , LiFe ₅ O ₈ ) and others, for biological and biomedical applications, iron oxide nanoparticles are the best choice due to their ease of synthesis, low toxicity, high saturation magnetization and susceptibility, biocompatibility, superparamagnetic action, and chemical stability [9]. To increase the stability of iron oxide MNPs, various types of coatings are used: inorganic compounds (silicon oxide, carbon, noble metals), synthetic (PEG, PVA, etc.) or natural (chitosan, polysaccharides, proteins, peptides ) polymers [10], while the scope of the obtained functionalized nanoparticles becomes much wider. Among various stabilizers or coating agents, various polysaccharides are often used due to their chemical and structural high sorption capacity and specific binding to the object to be isolated. Various magnetic nanoparticles carrying polysaccharides such as cellulose, chitosan, arabinogalactan, dextran, and amylose have been synthesized and functionalized with various biomolecules for use in biotechnological and biomedical research [11–13].

There is information in the literature about various methods of preparation, physicochemical properties, and the use of starch-activated MNPs.

The work [14] describes the preparation and properties of Fe ₃ O ₄ magnetic nanoparticles coated with starch. To obtain nanoparticles, the authors used the method of chemical precipitation from a solution of iron chlorides by adding an ammonia solution. Starch was immobilized on the surface by incubation of the synthesized nanoparticles in the prepared starch solution (60°C, 2 hours with vigorous stirring and 12 hours at room temperature). The described method makes it possible to obtain starch-coated magnetite nanoparticles of spherical shape and a diameter of about 20 nm. The authors showed that starch is immobilized on the surface of nanoparticles due to the interaction of its hydroxyl groups with iron, which ensures high colloidal stability of the resulting nanoparticles. Starch-coated magnetic nanoparticles exhibited superparamagnetic behavior with a blocking temperature of about 170 K and a saturation magnetization ranging from 30 to 50 emu/g.

The authors of [15] studied the structural properties of Fe ₃ O ₄ magnetic nanoparticles coated with starch of various origins (from corn and wheat). Using the described method of "green" synthesis of these magnetic nanocomposites (co-precipitation from solutions of iron salts FeSO ₄ and FeCl ₃ containing 0.5-2% starch in the presence of sodium hydroxide), spherical nanoparticles with an average size of 10 nm, having a high surface area and magnetization (40 emu/g). The work shows that the concentration of starch and sodium hydroxide affects the size of the synthesized particles, and that corn starch stabilizes magnetic nano-biocomposites to a greater extent, in contrast to starch obtained from wheat.

The disadvantages of the described materials is that they are not intended as sorbents for affinity chromatography of recombinant proteins.

The paper [16] describes a new approach to obtaining monodisperse starch-based magnetic polymer particles of controlled size, which can be used as a highly efficient material for immunomagnetic separation. The approach includes: 1) synthesis of Fe ₃ O ₄ nanoparticles coated with dextran (Dex-IONPs) by co-precipitation from an iron solution (FeSO ₄ and FeCl ₃ ) containing dextran with an ammonium peroxide solution during ultrasonic treatment; 2) two-stage treatment of starch with the enzyme pullulanase to form short-chain glucans (cleavage of amylopectin), the duration of the first stage is 4 hours, the second stage is overnight; 3) incubation of short-chain glucans with Dex-IONPs (24 hours), during which spontaneous formation of monodisperse magnetic polymer particles containing amylopectin occurs. To immobilize antibodies on the surface of the synthesized particles, the authors used a recombinant maltose-binding protein (MBP) genetically linked to the G protein of streptococcus (proteinG), which has the ability to bind Fc fragments of immunoglobulins. The efficiency of using the obtained particles for immunomagnetic separation was demonstrated using the isolation of E. coli O157:H7 cells as an example. The bound cells were eluted by adding a maltose solution.

The disadvantage of the described material is the complex method of obtaining, and the described material is not intended as a sorbent for affinity chromatography of recombinant proteins.

In [17], the issues of _the toxic effect of amylose-associated _Fe3O4 nanoparticles on the body of zebrafish, Danio rerio are considered in comparison with those not coated with carbohydrates. Since it has been shown that starch (and amylose) increases the biocompatibility of magnetic particles and can be used for various functionalization of the surface of nanocomposites with drugs, there are more and more works on the synthesis of magnetic starch-activated bionanocomposites suitable for drug delivery [18].

In [19], starch-stabilized magnetite iron oxide nanoparticles were obtained, having a quasi-spherical shape, with an average magnetic core size of 6–7 nm, an average hydrodynamic radius of 46 nm, and a saturation magnetization of 45 emu per 1 gram of particles. They were used for chemical conjugation with hepatocarcinoma-binding peptide. The authors studied the magnetic properties of the obtained composites and their specificity to human hepatocellular carcinoma cells in experiments in vitro and in vivo and showed that the obtained modified nanoparticles are a promising material for the diagnosis and therapy of malignant tumors (drug delivery, magnetic hyperthermia, and magnetic resonance imaging).

The disadvantages of the described materials is that they are not intended as sorbents for affinity chromatography of recombinant proteins.

In [20], spherical _Fe2O3 magnetic nanoparticles (90 nm in size) coated with starch were obtained _by coprecipitation, which was further modified with glutaraldehyde to immobilize the thermoalkaliphilic esterase enzyme to obtain a stable biocatalyst. The immobilization yield and esterase immobilization efficiency were 74% and 82%, respectively. In addition, the immobilization of esterase on the proposed particles improves the thermal stability of the enzyme.

Review [21] describes methods for obtaining magnetic composites (from nano to microspheres) based on starch and its various modifications and gives examples of their use as adsorbents for water purification from heavy metal pollution (Cd(II), Cr(VI), Cu (II)) and organic substances (drugs, oil, etc.).

The disadvantages of the described materials is that they are not intended as sorbents for affinity chromatography of recombinant proteins.

In [22], taken as a prototype, an enzymatic method for obtaining amylose-activated magnetic microspheres (size 2.09 ± 0.42 μm and magnetization 4.5 emu/g) was developed. The authors used the enzyme amylosucrose, which synthesizes amylose chains from sucrose, which are capable of self-organizing into spherical microstructures. It has been shown that magnetic iron oxide nanoparticles are effectively incorporated into amylose microspheres during their self-assembly; the reaction proceeds at 30°C for 24 hours. The resulting magnetic microspheres were used as an affinity sorbent for isolating green fluorescent protein (GFP) containing the MBP domain from the lysate of recombinant E. coli cells. The sorption capacity of the obtained microspheres was 72.31 μg of GFP per mg of microspheres. In the process of triple reuse, the sorption capacity of amylose microspheres was 88%.

The disadvantage of the prototype is the complex method of obtaining the material and its low specific sorption capacity.

The objective of the present invention is to obtain and study the properties of starch-activated magnetic iron oxide nanoparticles as an affinity sorbent for the isolation of recombinant proteins, including maltose-binding protein (MBP) as an auxiliary domain.

Technical result is to increase the specific sorption capacity of the magnetic affinity sorbent, which is starch-activated magnetic iron oxide nanoparticles.

The technical result is achieved by the fact that the magnetic affinity sorbent for the isolation of recombinant proteins is characterized by the fact that it consists of starch-activated magnetic iron oxide nanoparticles with an average size of 11.5 nm, a saturation magnetization value at room temperature of 29.8 emu/g, a blocking temperature close to to room temperature, one gram of which allows you to isolate 100-590 mg of recombinant MBP-containing hybrid proteins from the lysate of E. coli cells.

The defining differences of the proposed invention from the prototype are:

the resulting nanoparticles are characterized by significantly smaller sizes compared to the prototype - 11.5 nm versus 2.09 ± 0.42 μm, which provides a higher surface of interaction with molecules in solution;

the resulting nanoparticles have a saturation magnetization of 29.8 emu/g, and an average blocking temperature close to room temperature, which provides both a large magnetic response and colloidal stability;

the sorption capacity of the obtained affinity sorbent is 100-590 μg of recombinant MBP-containing hybrid proteins per 1 mg of nanoparticles, which exceeds the sorption capacity of the particles obtained in the prototype (72.31 μg of GFP per 1 mg of particles);

the purity of recombinant proteins purified using the obtained affinity sorbent is 80-94%;

the obtained starch-activated nanoparticles can be used three times without loss of their sorption capacity, and are also stable for up to 6 months when the samples are stored in a household refrigerator chamber (+8-12°C) with the addition of 0.05% NaN ₃ . Information about the stability of the material during storage in the prototype is not given;

a simple, cheap and convenient method for the synthesis of starch-activated iron oxide nanoparticles based on the co-precipitation method, in which starch is used as a stabilizing agent. The method does not require additional functionalization of the surface of nanoparticles and modification of starch;

The invention is illustrated by drawings.

Figure 1 shows images of starch-coated iron oxide nanoparticles obtained using a high-resolution transmission electron microscope.

On FIG. Figure 2 shows the Mössbauer spectra of starch-coated iron oxide nanoparticles obtained at room temperature.

On FIG. Figure 3 shows the IR spectra of iron oxide nanoparticles coated with starch. (a) - starch, (b) - iron oxide nanoparticles coated with starch

On FIG. 4 shows the temperature dependence of the coercive force (orange squares), fitting by equation (1) (black line) and the temperature dependence of the magnetization measured in a field of 5 kOe (blue squares).

On FIG. Figure 5 shows 12.5% SDS-PAGE electropherograms of protein preparations during the purification of MBP-TnI (A), MBP-MIA (B) and MBP-Surv (C) using the obtained starch-activated nanoparticles: proteins of transformed E. coli cells up to (columns 1) and after (columns 2) IPTG induction; cytoplasmic fractions (columns 3); fractions after elution with a solution of 10 mm maltose (columns 4); standard proteins (BioRad, USA) (columns 5), the molecular weights of which are shown by numbers on the right. The arrows indicate the bands of the fusion proteins.

The essence of the invention lies in the synthesis of starch-activated magnetic iron oxide nanoparticles, which are used as a sorbent for the affinity isolation of recombinant proteins having a maltose-binding protein (MBP) domain as an auxiliary fragment.

An example of the synthesis of starch-activated magnetic iron oxide nanoparticles:

Iron oxide nanoparticles are obtained by co-precipitation from a solution of the following composition: Mohr's salt - 50 g/l, Na citrate - 50-100 g/l, EDTA-Na2 - 20 g/l, starch - 5-50 g/l. At a temperature of 80°C, NaOH (0.1 M) was added to the solution until a neutral pH was reached. The required temperature is maintained using a water thermostat. Coated iron oxide nanoparticles are thoroughly washed with distilled water to remove ions.

Physical properties of starch-activated magnetic nanoparticles:

Electron microscopic studies are performed on a Hitachi HT7700 transmission electron microscope at an accelerating voltage of 100 kV (Fig. 1). The particles are cubic nanocrystals with an average size of 11.5 nm. The diffraction pattern of the studied sample is typical for the structure of magnetite or maghemite.

Mössbauer spectra are measured on an MS-1104Em spectrometer with a ⁵⁷ Co(Cr) source at room temperature (Fig. 2). The experimental spectrum is well described by the components shown in the figure. Fit error 3%. 4 sextets and one doublet are found. The results of the decoding indicate that all iron ions are in the trivalent state. Thus, the sample can be considered defective maghemite γ-Fe ₂ O ₃ .

Table 1 presents the results of the interpretation of the Mössbauer spectra.

Table 1 IS H QS W ₃₄ A 1 0.34 474 0.01 0.50 0.14 S1 0.42 440 -0.04 0.73 0.24 S2 0.45 386 0.01 0.67 0.26 S3 0.37 193 0.01 1.24 0.25 S4 0.35 - 0.96 1.08 0.11 D

IS - isomeric chemical shift with respect to bcc-Fe, QS - quadrupole splitting,

W is the width of the absorption line, Η is the hyperfine field on the iron nucleus, A is the fractional position occupancy.

IR absorption spectra of the studied samples are obtained in a KBr matrix on a vacuum Fourier-IR spectrometer VERTRX-80V (BRUKER) in the spectral range of 7500 - 380 cm ^-1 with a spectral resolution of 0.2 cm ^-1 (Fig. 3). It should be noted that the spectra of compounds with magnetic nanoparticles have a characteristic increase in the background in the region of 2000-4000 cm ^-1 . In the spectrum (Fig. 3b) bonds in the region of 1200-1500 cm ^-1 disappear, there are bonds in the region of 800-1200 cm ^-1 and hydroxyl. Spectral analysis shows: absorption peaks at 388 and 570 cm ^-1 in the spectrum of starch-coated magnetic nanoparticles refer to O-Fe-O bending vibrations and Fe-O stretching modes, starch chemisorption occurs on the surface of nanoparticles through acetal bonds.

Table 2 shows the IR absorption peaks and their correlation.

table 2 Range maximum absorption band,
starch Absorption band maximum, starch +MNPs description 388 O-Fe-O 400-800 413 Range of vibrations of the pyranose ring and δ-hydroxyl groups 432 487 527 573 corresponds to the vibrations of the chain C - C - C ... - 570 Fe-O 617 Range of oscillations of the pyranose ring and δ hydroxyl groups 706 767 800-1000 855 S-O to S-O-N 866 922 900 1000-1200 1001 C-O stretching
internal vibrations of C-O bonds (Characteristic bands for polysaccharides due to the presence of acetal bonds) 1025 1075 1092 1150 1161 1200-1500 1238 δ - CH ₂ groups in CH ₂ OH 1341 δ - O-H bonds in CH ₂ OH 1368 δ - bonds of CH ₂ groups 1421 δ - CH ₂ groups 1461 δ-OH 1500-2000 1654 1635 δ - bonds in H-O-H (adsorbed water) 2000-3000 2060 ν - bonds in CH and CH ₂ groups 2153 2890 2930 2928 S-N 3000-4000 3406 3413 Internal vibrations of OH groups involved in intermolecular and intramolecular H-bonds

In order to determine the magnetic characteristics (magnetization, coercive force, blocking temperature) of nanoparticles, hysteresis loops are recorded in fields from -2 kOe to 2 kOe in a temperature range of 80–295 K. The saturation magnetization of nanoparticles at room temperature is 29.8 emu/g.

The blocking temperature is an important characteristic for biomedical applications of nanoparticles, in particular magnetic separation. The blocking temperature is the temperature separating the superparamagnetic state from the blocked state. Superparamagnetic nanoparticles are characterized by hysteresis-free behavior and better colloidal stability. Nanoparticles in the blocked state are characterized by field hysteresis and higher magnetic susceptibility, but they are less stable in a colloid. Nanoparticles with a blocking temperature close to room temperature are more preferable for use in magnetic separation.

может быть описана уравнением (1), согласно которому коэрцитивная сила

уменьшается с температурой

вплоть до температуры блокировки

[23]. Данное уравнение обычно используется для однодоменных невзаимодействующих наночастиц при температурах, ниже температуры блокировки

[24].Temperature dependence of the coercive force

can be described by equation (1), according to which the coercive force

decreases with temperature

up to blocking temperature

[23]. This equation is usually used for single-domain non-interacting nanoparticles at temperatures below the blocking temperature

[24].

(коэрцитивное поле при T=0K),

,

(показатель степени) являются подгоночными параметрами.Where

(coercive field at T=0K),

,

(exponent) are adjustable parameters.

. Видно, что при комнатной температуре экспериментальные значения

существенно снижаются, свидетельствуя о близости температуры блокировки к комнатной температуре. Подгонка экспериментальных данных уравнением (1) дает следующие параметры подгонки:

,

,

.On FIG. 4 shows the experimental temperature dependence of the coercive force

. It can be seen that at room temperature the experimental values

decrease significantly, indicating that the blocking temperature is close to room temperature. Fitting the experimental data with equation (1) gives the following fitting parameters:

,

,

.

Thus, the average blocking temperature is close to room temperature and therefore such nanoparticles are well suited for use in magnetic separation.

Use of starch-activated magnetic nanoparticles for one-step affinity purification of recombinant proteins:

The resulting starch-activated magnetic nanoparticles were used as an affinity sorbent for the purification of the following recombinant MBP-containing fusion proteins: cardiac troponin I (MBP-cTnI), melanoma inhibitory activity protein (MBP-MIA), and survivin (MBP-Surv).

The expression of the hybrid proteins MBP-cTnI, MBP-MIA and MBP-Surv is carried out in E. coli BL21-CodonPlus (DE3)-RIPL cells transformed with the corresponding plasmids. Cells are cultured in LB medium containing 2 g/l glucose and 200 µg/ml ampicillin at 37°C until an optical density of OD ₅₉₀ = 0.5-0.7 is reached. Induction of protein synthesis is carried out by adding IPTG (1 mm), the cells are cultured for another three hours, and then precipitated by centrifugation (4000 g, 20 min, 4°C), the supernatant is discarded.

Cell biomass is resuspended in buffer A (20 mM Tris-HClpH 7.5, 0.2 MNaCl, 1 mM EDTA) at a ratio of 1:5 (w/v), sonicated (6 times 20 sec) at 0°C and centrifuged again. The precipitate is discarded, the supernatant (cytoplasmic fraction) (1 ml) is mixed with 0.5 ml suspension of starch-activated nanoparticles (in buffer A) (20 mg/ml) and incubated at 4°C for one hour with stirring. Then the nanoparticles are fixed with a magnet, and the solution is removed by pipetting. After washing the nanoparticles (three times with 1 ml buffer A), the fusion protein is eluted with a buffer containing 10 mM maltose, 20 mM Tris-HClpH 7.5, 0.2 M NaCl, 1 mM EDTA (elution buffer).

The purity of protein preparations during isolation is controlled by electrophoresis in 12.5% polyacrylamide gel (PAAG) containing 0.1% SDS according to the Laemmli method [25]. Protein concentration is determined spectrophotometrically using the DC™ kit ProteinAssay (BioRad, USA). Bovine serum albumin is used as a calibration protein.

Reuse of starch-activated magnetic nanoparticles.

0.5 ml suspension of starch-activated magnetic nanoparticles (20 mg/ml) is used to purify the MBP-cTnI fusion protein three times in succession. After applying the cytoplasmic fraction (0.8 ml), washing the nanoparticles and eluting the protein (as described in example 3), the nanoparticles are washed 5 times with elution buffer, equilibrated with buffer A and reused to purify the protein from the next portion of the lysate.

The yield of purified MBP-cTnI is 94.7, 104.1 and 102.8 μg per milligram of starch-activated MNPs after the first, second and third isolation cycles, respectively.

Thus, the proposed affinity sorbent based on starch-activated magnetic iron oxide nanoparticles has the following physical characteristics: average size 11.5 nm, saturation magnetization value at room temperature 29.8 emu/g, blocking temperature close to room temperature (377 K) .

The sorbent has a high sorption capacity - 100-590 mg of recombinant MBP-containing hybrid proteins per gram of nanoparticles and stability, makes it possible to obtain high-purity protein preparations (80-94%) in one stage of chromatographic purification, and can also be used three times without loss sorption capacity.

The method for obtaining magnetic nanoparticles is highly reproducible (the coefficient of variation is 14.2%).

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Claims (1)

A magnetic affinity sorbent for the isolation of recombinant proteins, characterized in that it consists of starch-activated magnetic iron oxide nanoparticles with an average size of 11.5 nm, a saturation magnetization value at room temperature of 29.8 emu/g, a blocking temperature close to room temperature, one gram of which allows you to isolate 100-590 mg of recombinant MBP-containing hybrid proteins from the lysate of E. coli cells.

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