Method for purifying recombinant proteins using ice-binding domain

Abstract

The present invention relates to a method for purifying a recombinant protein using an ice-binding domain. The ice-binding domain of the present invention has characteristics of binding to various crystal lattice planes and exhibits high activity, high solubility, and high expression levels, thereby being able to be advantageously used for purifying a target protein.

Description

Recombinant protein purification method using ice-binding domains

[0001] The present invention relates to a method for purifying recombinant proteins using the ice-binding properties of an ice-binding domain.

[0002] Ice-binding proteins (IBPs) refer to proteins that bind to specific surfaces of ice to inhibit or promote its growth. Ice-binding proteins were first discovered in the 1960s in Antarctic cod (Notothenioid) inhabiting the Southern Ocean and South Atlantic, and are known as proteins that represent the cold adaptation mechanism of polar organisms, as they help prevent fish serum from freezing even at temperatures as low as -1.87°C. These ice-binding proteins were initially named antifreeze glycoproteins (AFGPs), and subsequently, ice-binding proteins existing in various forms were found not only in fish but also in plants, insects, and microorganisms.

[0003] Ice-binding proteins possess unique properties that enable them to bind to ice. It is generally known that specific parts of ice-binding proteins bind to ice, and these are referred to as ice-binding sites (IBS). These IBSs are typically composed of hydrophilic residues, with threonine or serine present in particularly high proportions. These amino acids are known to interact with ice through hydrogen bonding with water molecules present in the ice. Furthermore, methyl groups on threonine residues are also known to play a crucial role in ice binding. Through hydrogen interactions with water molecules, methyl groups induce an aligned arrangement of water molecules, and these aligned water molecules are known to interact with water molecules in the ice. This alignment of water molecules leads to the formation of a hydration layer, in which water molecules interact with water molecules in the ice.

[0004] The activity of ice-binding proteins is measured through thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activities. TH activity is a method that measures the difference between the freezing point and the melting point, and proteins are classified into moderately active proteins and hyperactive proteins based on the magnitude of activity. Proteins with activity below 0.5°C belong to the moderately active protein group, while those with activity above 2.5°C belong to the hyperactive protein group. Previous studies have indicated that repetitive sequences present in the fusion binding site of hyperactive proteins act as binding motifs, and that this region binds to various crystal lattice planes of ice. Hyperactive proteins maintain high activity by binding to these various crystal lattice planes of ice.

[0005] Purification is an essential step for obtaining pure proteins to analyze or study their properties. Generally, to obtain a desired protein (target protein), genetically engineered E. coli and corresponding vectors are used to induce gene transcription and translation, thereby maximizing protein expression. Since the expressed protein exists alongside proteins necessary for the survival of the E. coli, it undergoes a process of isolation and purification.

[0006] A common method used to purify recombinant proteins is the Ni-NTA purification system. This system utilizes the binding properties of nickel or cobalt with a His-Tag attached to the N- or C-terminus of a protein molecule to achieve separation and purification. In this method, proteins are expressed and extracted from host cells, and then a protein solution containing the His-Tag is passed through a Ni-NTA (nickel-bound agarose) resin. In this manner, proteins with the His-Tag bind to the resin, while E. coli-derived proteins without the His-Tag are washed away. Finally, the proteins with the His-Tag are eluted from the resin to obtain the purified protein. This method is relatively simple, efficient, and one of the well-known techniques capable of purifying proteins to high purity. However, because this method uses nickel during purification, using it for the purification of metal-binding proteins poses a high risk of causing problems during protein characterization studies, as the nickel can affect the proteins. Furthermore, even if a protein is not a metalloprotein, it tends to bind to ionic metals, so the binding of nickel during purification often induces protein denaturation or causes polymer aggregation.

[0007] The second common method for purifying recombinant proteins is Size Exclusion Chromatography (SEC). Size exclusion chromatography is a chromatographic technique that separates molecules based on size; it is used to separate and purify proteins, nucleic acids, and macromolecules, and is also a useful tool for determining the molecular weight distribution of macromolecules or obtaining purified fractions of biomolecules. SEC utilizes a column filled with a stationary phase having different pore sizes; when proteins are injected into the column, small molecules can pass through the pores of the stationary phase, while large molecules cannot enter and flow out. Consequently, larger proteins elute first, followed by smaller proteins, thus separating them according to size. However, this method presents difficulties in purification if the target protein is similar in size to an undesirable protein, and it requires expensive specialized equipment for the column and solution flow.

[0008] Accordingly, the inventors devised a metal-free method to overcome the disadvantages of heavy metal ion-mediated purification methods found in some existing purification methods. As a result of their efforts to establish a system capable of large-scale purification, they invented an ice-binding domain-mediated recombinant protein purification system and method applicable to the purification of a wide range of recombinant proteins. This method purifies target proteins by utilizing the ice-binding properties of the ice-binding domain; because it uses ice, it is cost-effective, environmentally friendly, and offers excellent scalability, making it suitable for large-scale protein purification.

[0009] The object of the present invention is to provide a novel ice-binding domain.

[0010] Another object of the present invention is to provide a fusion protein comprising the ice-binding domain and the target protein, a polynucleotide encoding the fusion protein, an expression vector comprising the polynucleotide, and a host cell transformed with the expression vector.

[0011] Another objective of the present invention is to provide a method for purifying a recombinant protein using a fusion protein comprising the ice-binding domain and the target protein.

[0012] In order to achieve the above objective,

[0013] The present invention provides an ice-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 4.

[0014] In addition, the present invention provides a fusion protein comprising the ice-binding domain and the target protein, a polynucleotide encoding the fusion protein, an expression vector comprising the polynucleotide, and a host cell transformed with the expression vector.

[0015] In addition, the present invention provides a method for purifying a recombinant protein using a fusion protein comprising the ice-binding domain and the target protein.

[0016] The present invention relates to a method for purifying recombinant proteins using an ice-binding domain. The ice-binding domain of the present invention has the characteristic of binding to various crystal lattice planes and possesses high activity, high solubility, and high expression levels, making it useful for purifying target proteins. Furthermore, since the present invention purifies target proteins using ice as a medium, it is cost-effective, environmentally friendly, and has good scalability, making it suitable for large-scale purification of target proteins.

[0017] Figure 1a is a flowchart illustrating the process for identifying an ice-binding domain suitable as a tag protein in a protein purification system using an ice-binding domain.

[0018] Figure 1b is a figure showing the results of identifying ice-binding proteins from the method of Figure 1a.

[0019] Figure 1c is a figure showing the domain structure of the identified ice-binding protein.

[0020] Figure 2a shows the results of confirming the temperature history (TH) activity of the ice binding domain.

[0021] Figure 2b is a figure showing the results of analyzing the ice binding domain and the binding surface of ice.

[0022] Figure 3a shows the results of confirming the ice recrystallization inhibition (IRI) activity of the ice binding domain.

[0023] Figure 3b shows the results of observing the recrystallization of the ice binding domain.

[0024] Figure 4a shows the results of analyzing the melting temperature (Tm) of the ice binding domain.

[0025] Figure 4b shows the results of comparing the sequences of ice binding domains using the Multiple Sequence Alignment (MSA) method.

[0026] Figure 4c is a figure showing the results of analyzing the crystal structure of the ice binding domain.

[0027] Figure 4d is a structural diagram showing three amino acid residues (Met74, Ala88, Ala97) associated with the thermal stability of the ice-binding domain.

[0028] Figure 4e shows the results of analyzing the melting temperature (Tm) of the ice-binding domain variant.

[0029] Figure 5a is a figure showing an apparatus used in a protein purification system using an ice-binding domain.

[0030] Figure 5b shows the results of analyzing a protein purified using an ice-binding domain and then analyzed by SDS-PAGE.

[0031] Figure 5c is a graph showing the purity and recovery rate of a protein purified using an ice-binding domain.

[0032] Figure 6a is a photograph showing the protein binding site and distribution by vertically cutting the binding plane of the ice binding domain.

[0033] Figure 6b shows the results of observing the binding surface of the ice binding domain using a confocal microscope.

[0034] Figure 6c shows the results of observing the binding surface of the ice binding domain according to the depth of the ice surface using a confocal microscope.

[0035] Figure 7 shows the results of analyzing the purity and recovery rate of purified protein using additives (5% glycerol, 1 mM EDTA, 1 mM DTT) used in protein purification.

[0036] Figure 8a shows the results of analyzing the solubility of the ice-binding domain.

[0037] Figure 8b shows the results of analyzing the expression level of the ice binding domain.

[0038] FIG. 9 is a figure showing a recombinant protein purification system using the ice binding domain of the present invention.

[0039] The present invention will be described in detail below.

[0040] The present invention provides an ice-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 4.

[0041] In order to develop a new protein purification method using an ice medium by utilizing the ice affinity of ice-binding domains, the inventors identified three new DUF3494 ice-binding domains (CoIBD1; SEQ ID NO. 1, CoIBD2; SEQ ID NO. 2, CoIBD3; SEQ ID NO. 3) from Candidatus Cryosericum odellii SMC5 in a protein database and confirmed the potential for their use in the purification of recombinant proteins by confirming that said ice-binding domains exhibit hyperthermal hysteresis activity and ice recrystallization inhibition (IRI) activity. In addition, by analyzing high-resolution X-ray structures and nucleotide sequences, an ice-binding domain variant (eCoIBD1; SEQ ID NO. 4) was produced and confirmed to have improved thermal and chemical stability without losing ice-binding properties. Therefore, the ice-binding domain of the present invention may include or be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs. 1 to 4.

[0042] In addition, the present invention provides a fusion protein comprising the ice-binding domain and the target protein.

[0043] In the present invention, the ice-binding domain may be connected to either the N-terminus or the C-terminus, as long as it does not substantially affect the functionality of the target protein. Here, not substantially affecting the functionality of the protein means that the protein maintains at least 80%, preferably at least 95%, of its activity before fusing the ice-binding domain.

[0044] In the present invention, “target protein” refers to any protein intended for production or purification, including peptides. Green fluorescent protein (GFP) has been used as an example of the target protein, but is not limited thereto. The term “target protein” refers to a protein intended to be produced by the biotechnological method according to the present invention, and is not specifically limited to any one of them; preferably, it may include a protein that can be used for medical, industrial, diagnostic, experimental, etc.

[0045] At this time, the ice-binding domain and the target protein may come into direct contact or be mediated by a mediator such as a linker, and the linker may be designed to be cleavable after immobilization so that the target protein can be recovered from the fusion protein.

[0046] In the present invention, the fusion protein may further comprise a linker sequence containing a cleavage site of the protein cleavage enzyme enterokinase (EC 3.4.21.9) between the ice-binding domain and the target protein. The cleavage site of enterokinase may be included between the affinity protein tag and the target protein, and may be cleaved by the enzyme to separate the affinity protein tag and the target protein. The cleavage site of the protein cleavage enzyme consists of the amino acid sequence DDDDK (Asp-Asp-Asp-Asp-Lys, SEQ ID NO. 9) and was used to remove the affinity protein tag, but is not limited thereto and may include a cleavage site generally used for protein purification.

[0047] In the present invention, the ice binding domain may be encoded by a nucleotide sequence consisting of SEQ ID NOs 5 to 8.

[0048] In addition, the present invention provides a polynucleotide encoding the fusion protein.

[0049] In the present invention, the fusion protein is characterized by comprising an ice-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 4. The amino acid of SEQ ID NO 1 may be encoded by the polynucleotide sequence of SEQ ID NO 5, the amino acid of SEQ ID NO 2 may be encoded by the nucleotide sequence of SEQ ID NO 6, the amino acid of SEQ ID NO 4 may be encoded by the polynucleotide sequence of SEQ ID NO 7, and the amino acid of SEQ ID NO 4 may be encoded by the nucleotide sequence of SEQ ID NO 8.

[0050] In the present invention, the polynucleotide encoding the fusion protein may include a linker sequence and a cleavage site of a protein cleavage enzyme between the ice-binding domain and the target protein, and a multiple cloning site sequence capable of inserting the target protein.

[0051] In addition, the present invention provides an expression vector comprising the polynucleotide.

[0052] In the present invention, an expression vector refers to a vector capable of expressing a target protein or nucleic acid (RNA) in a suitable host cell, and comprises a gene construct that includes an essential regulatory element operably linked to enable the expression of a polynucleotide (gene) insert.

[0053] In the present invention, “operably linked” means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function, and are linked so that a gene can be expressed by the expression control sequence. The expression control sequence refers to a DNA sequence that regulates the expression of an operantly linked polynucleotide sequence in a specific host cell. Such a control sequence includes a promoter for carrying out transcription, any operator sequence for regulating transcription, a sequence coding for a suitable mRNA ribosome binding site, a sequence regulating the termination of transcription and translation, a start codon, a stop codon, a polyadenylation signal, and an enhancer.

[0054] In the present invention, the expression vector is not particularly limited in type as long as it is a vector commonly used in the field of cloning, and examples include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and virus vectors. The plasmids include plasmids derived from Escherichia coli (pBR322, pBR325, pUC118 and pUC119, pET-22(+)), plasmids derived from Bacillus subtilis (pUB110 and pTP5), and plasmids derived from yeast (pPICZ, YEp13, YEp24 and YCp50), and the viruses may include animal viruses such as retroviruses, adenoviruses, or vaccinia viruses, insect viruses such as baculoviruses, and preferably, the pET-28a(+) vector may be used.

[0055] In the present invention, the expression vector may be transformed by introducing it into a cell to produce a target protein or a fragment thereof by methods known in the art, such as, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and known methods for introducing nucleic acids into a cell. Cells transformed with the expression vector according to the present invention may overexpress or produce the fusion protein according to the present invention in large quantities.

[0056] In the present invention, the expression vector may be used in combination with a recombinant expression vector.

[0057] In the present invention, recombination can be used interchangeably with genetic manipulation and means producing a form of gene that does not exist in nature by using molecular cloning experimental techniques such as modifying, cutting, and linking genes.

[0058] In the present invention, expression means the production of a protein or nucleic acid in a cell.

[0059] In addition, the present invention provides a host cell transformed with the expression vector.

[0060] In the present invention, "transformation" refers to a phenomenon in which external DNA is introduced into a cell to artificially induce genetic changes, such as by introducing DNA into a host so that the DNA becomes capable of replication as a chromosomal factor or through the completion of chromosomal integration. The above-mentioned transformation method may be performed by methods known in the art.

[0061] In the present invention, the type of host cell is not particularly limited as long as it is a cell that can be used to express a polynucleotide included in the expression vector of the present invention. The cell (host cell) transformed with the expression vector according to the present invention may be a prokaryote (e.g., Escherichia coli), a eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), an animal cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, insect cells, or a hybridoma derived therefrom), and preferably may be Escherichia coli (E. coli), but is not limited thereto.

[0062] In addition, the present invention provides a method for purifying a recombinant protein using a fusion protein comprising the ice-binding domain and the target protein.

[0063] In the present invention, the details regarding the ice-binding domain and the target protein are as described above.

[0064] In the present invention, the method may include: (1) a step of preparing a solution comprising a fusion protein comprising the ice-binding domain of claim 1 and a target protein; (2) a step of introducing the solution into a container containing ice to induce adsorption of the recombinant protein; and (3) a step of separating the target protein from the fusion protein.

[0065] In the present invention, the above step (2) may be performed under conditions of 0°C or lower, preferably -2°C, but is not limited thereto.

[0066] In the present invention, step (2) can be performed by a water chiller circulator. For the protein purification system, a device combining a water chiller circulator and a bottle rotation system was used.

[0067] In the present invention, the method for purifying the target protein may be performed by treatment with a protein cutting enzyme included in the fusion protein.

[0068] The present invention will be explained in more detail below through examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0069] Experimental method

[0070] Cloning, expression, and purification of the ice-binding domain

[0071] The ice-binding domains (CoIBD 1, 2, 3) were selectively amplified by performing Polymerase Chain Reaction (PCR) using the CoIBP 1, 2, and 3 genes as templates. The amplified DNA fragments were inserted into the NdeI restriction enzyme site of the pET-28a(+) plasmid (Novagen) using the ligand-independent cloning method. Consequently, a recombinant plasmid was generated with six histidine residues attached to the N-terminus of the domains for expression and purification. The plasmid and recombinant protein expression were maintained and amplified using Escherichia coli DH5α and BL21 (DE3) (Enzynomics), respectively. When the optical density of the growing cells reached 0.5 at 600 nm, the expression of the recombinant protein was induced using 1.0 mM IPTG (isopropyl β-D-1-thiogalactopyranoside). Cells were obtained by centrifuging at 5,000xg for 20 minutes after culturing at 15℃ for 20 hours.

[0072] The cells were resuspended and disrupted by sonication, after which the cell debris was centrifuged at 15,000 xg for 40 minutes at 4°C. The supernatant was loaded onto a Ni-NTA agarose column (Qiagen). The elution fraction containing His-tagged proteins was collected and concentrated, and N-terminal His-tagged cleavage was performed by treatment with thrombin. Next, the solution was loaded onto a Superdex 200 prep-grade column (GE Healthcare) to remove thrombin and other contaminants. The purified proteins were concentrated to 97 mg / mL for CoIBD1, 98 mg / mL for CoIBD2, and 91 mg / mL for CoIBD3 in a buffer supplemented with 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 5% glycerol using an Amicon Ultra Centrifugal Filter (Merck).

[0073] Size exclusion chromatography (SEC)

[0074] SEC was performed to evaluate the oligomerization of CoIBD. A Superdex 200 10 / 300 GL column was stabilized with 20 mM Tris-HCl (pH 8.0) and 200 mM NaCl. Molecular weight standard curves were obtained using a 15,600 kDa protein standard mixture (Sigma-Aldrich). This mixture included ribonuclease A (13.7 kDa), ovalbumin (44.3 kDa), gamma globulin (150 kDa), and thyroglobulin (669 kDa). Protein samples were diluted to 1 mg / mL in the same equilibration buffer and loaded onto the column at a flow rate of 0.4 mL / min at 20°C.

[0075] Crystallization and Data Collection

[0076] Samples were screened on 96-well sitting drop plates (Emerald Bio) at 20°C. A mosquito high-throughput crystallization robot (TTP Labtech) was used to verify initial crystallization conditions. Crystals were grown in 35% (w / v) PEG 4000 for CoIBD1, 22% PEG 8000, 0.1 M sodium cacodilate (pH 5.0), and 0.2 M sodium acetate trihydrate for CoIBD2, and 3.5 M sodium formate (pH 7.0) for CoIBD3. X-ray diffraction datasets were collected from beamlines 5C and 7A of the Pohang Light Source operated by the Pohang Accelerator Laboratory.

[0077] Determination and Improvement of Protein Structure

[0078] X-ray diffraction data were processed, integrated, and scaled using HKL-2000 or XDS. Molecular substitutions were performed using MOLREP integrated into CCP4i. After manual correction and refinement using Coot, the entire structure was refined using REFMAC5 refinement. All data were validated using MolProbity and stored in the Protein Data Bank.

[0079] The MOLREP program from the CCP4i family was used to determine the crystal structure of proteins via molecular substitution methods, utilizing an alphafold model as the search model. The structures were purified using REFMAC5 and phenix.refine. MolProbity was used to verify model quality, and all structural representations were generated using PyMOL. Mutation coordinates and structural factors were stored in the protein data bank RCSB.

[0080] Temperature history activity (TH activity) analysis

[0081] Approximately 1.5 μL of buffer or a sample containing an ice-binding domain was placed in a glass coverslip and placed on a Linkam TMHS6000 cold stage (Linkam Scientific Instruments). The temperature was rapidly decreased to -25°C at a rate of 60°C / min and then slowly increased until a single ice crystal remained. The Tm of the sample was observed. The sample was incubated at Tm = -0.1°C for 5 minutes, and then the temperature was lowered at a rate of 0.1°C / min. The freezing point (Tf) of the sample was recorded as the ice crystal began to grow. The TH value was calculated using the following formula: TH = |Tm - Tf|. Tm and Tf values ​​were measured three times, and data were expressed as mean ± SD. Analysis was performed using an Olympus BX51 microscope through a cross-section of Polaroids, and images were captured using an Olympus DP71 CCD camera.

[0082] Ice-recrystalization inhibition (IRI) activity analysis

[0083] IRI was measured by modifying the “splat cooling” method. Domain samples were diluted to the desired concentration in Tris buffer (20 mM Tris-HCl (pH 8.0), 200 mM NaCl). A 10 μL droplet was dropped 1.20 m onto a 16 mm glass coverslip placed on polished aluminum in liquid nitrogen. Upon impact with the cold coverslip, the droplet immediately froze into a wafer approximately 12 mm in diameter and 10 μm thick. The coverslip was transferred to a Linkam TMHS6000 cold stage (Linkam Scientific Instruments) and maintained at -6°C for 30 minutes. Analysis was performed using an Olympus BX51 microscope through crossed Polaroids, and images were captured using an Olympus DP71 CCD camera. The largest particle size along the axis was measured at various viewing angles. The maximum average particle size was calculated using the average of the 30 largest ice particles. IRI was calculated by dividing the average crystal size containing the domain by the average crystal size not containing the protein.

[0084] Fluorescence-based ice plane affinity (FIPA)

[0085] To elucidate the mechanism of CoIBD binding to ice, the FIPA assay was performed using fluorescently labeled ice-binding domain samples. Type III AFP was used as a control. To produce the sfGFP-IBD fusion protein, the gene encoding sfGFP containing the Gly-Gly-Ala-Gly linker was cloned into the pET-28a(+) vector at the NdeI site. The IBP sequence was amplified and replicated into the pET-28a(+)-sfGFP fusion vector between the EcoRI and SacI restriction enzyme sites. The resulting plasmid expressed the His-tag-sfGFP-IBD fusion protein. The fusion protein was then expressed and purified following the same procedure used for the wild-type protein.

[0086] A single ice crystal was identified between two cross-polarizing filters. After determining the c-axis, the macroscopic single ice crystal was mounted on the cold finger with the basal plane orientation perpendicular to the major axis of the cold finger. The ice crystal was then grown to a diameter of 4 cm in filtered deionized water at -7°C to form a hemispherical shape. Next, the ice hemisphere was transferred to a fluorescent protein solution (0.1 mg / mL) and grown to a diameter of 5 cm. The combined ice crystal was removed from the cold finger and stored at -20°C for 1 hour before visualization. Subsequently, the ice crystal was transferred to a refrigerator at -10°C and excited using light of 460–500 nm. Images were captured using a fluorescent filter.

[0087] Site-directed mutation induction

[0088] Site-directed mutation induction was performed to introduce a single amino acid substitution into the desired coding region of IBD. The nucleotide sequences of the variants were verified via DNA sequencing. All mutants were expressed and purified using the same method as that used for the wild-type domain.

[0089] Thermal shift assay

[0090] To evaluate the thermal and chemical stability of CoIBD, heat transfer analysis was performed using various solvents (DMSO, glycerol, methanol, ethanol, ethylene glycol, propylene glycol, and urea). These substances were dissolved in buffers supplemented with 20 mM Tris-HCl (pH 8.0) and 200 mM NaCl at concentrations of 5%, 10%, 15%, and 30% (v / v) for the solvents, and 1–4 M for the urea. To assess the stability of CoIBD, 10 μM of the domain was added to each mixture. Heat transfer analysis was performed using a Tycho NT.6 instrument (NanoTemper Technologies GmbH). Samples were placed in glass capillaries and heated from 35°C to 95°C at a rate of 30°C / min. The ratio of fluorescence signals at 350 / 330 nm and the melting temperature of the domain samples were determined by internal evaluation using the Tycho machine. All experiments were repeated three times.

[0091] Circular dichroism spectroscopy

[0092] Circular dichroism spectroscopy was performed using a Chirascan CD spectrometer (Applied Photophysics Co.) and a Peltier temperature controller. Far-UV CD measurements were taken at 20°C from 190 to 260 nm using 1 mm path length quartz cuvettes (Hellma). A protein concentration of 5 μM was used to determine the composition of the secondary structure. The buffer spectrum was subtracted, and the three spectra were averaged to obtain the final trace. All measurements were acquired at 10°C using 1 mm path length cells at 0.5 nm intervals within the wavelength range of 190 to 260 nm. The data were calculated using the mean residual ellipsity (mdeg cm⁻¹). 2 dmol -1 It is displayed as ).

[0093] Ice affinity purification (IAP) system using ice binding domains

[0094] The experimental equipment used a water chiller circulator containing an antifreeze solution composed mainly of ethylene glycol and a rotary motor connected to an assembly in which two long cylindrical metal rods were arranged parallel to each other and tilted at an angle of about 20°.

[0095] To form an initial ice crust, a 500 mL Scott bottle containing 150 mL of water was manually rotated in dry ice for 5 to 10 minutes. Subsequently, approximately 20 to 30 mL of water was used to form a thin layer of ice on the inner surface of the bottle, and the remaining unfrozen water was removed. The bottle was then manually rotated in the dry ice until freezing was complete and visible cracks appeared at the bottom of the ice. Next, 140 mL of the sample was filled into the frozen bottle and attached to the equipment. For protein purification, the motorized equipment containing the bottle was immersed in a liquid bath. The equipment was continuously rotated at approximately 4.5 rpm, and the bath temperature was maintained at -2°C during the experiment. All purification experiments were performed at 4°C.

[0096] Samples containing eCoIBD-SfGFP were pre-cooled to prevent the initial ice base from melting. Approximately 120–140 mL of sample is sufficient for a 500 mL Scott vial, and this ratio can be adjusted depending on the vial size. The vial was rotated in a water bath for about 1 hour to 1 hour and 30 minutes to freeze 40–50% of the sample volume. Then, the unfrozen solution was removed, and the vial was rotated for 2 minutes with 120 mL of pre-chilled water to rinse away non-specific proteins. The frozen samples were thawed and collected. The samples thawed from the initial purification were further purified to increase both purity and protein recovery rate, thereby optimizing process efficiency.

[0097] Quantitative analysis of protein expression

[0098] Single E. coli BL21(DE3) colonies were treated with kanamycin 50 μg-mL -1 The cells were cultured overnight in this supplemented LB medium. Subsequently, using the overnight culture results, 100 mL of freshly prepared kanamycin-supplemented LB medium was inoculated at a 1:100 dilution. When an optical density of 0.6–0.65 was reached at 37°C, IPTG at final concentrations of 0.1 mM, 0.5 mM, and 1 mM was added to the LB medium. Each concentration was distributed into three flasks. The culture medium was then incubated at 15°C for 3, 8, and 22 hours, after which cells were harvested by centrifugation and pellets were collected at each time point. For the condition without IPTG induction, cells were collected after 3 hours following the same procedure. The cell pellets containing eCoIBD1 were resuspended in Buffer A and sonicated over ice. After centrifugation at 10,000×g for 30 minutes at 4°C, the supernatant was collected. Next, 15 μL of sample was extracted for SDS-PAGE analysis. eCoIBD1 expression was quantified by scanning the gel using a Gel Doc EZ Imager (Bio-Rad). Subsequent analysis was performed using ImageLab version 3.0, built 11 (Bio-Rad). The intensity of the eCoIBD1 band was determined using volume intensity measurements. To express the relative expression level as a percentage, this value was normalized to the highest volume intensity set to 100%.

[0099] Protein Solubility Analysis

[0100] The protein was diluted with a series of buffers to a final volume of 100 μL in a 1.5 mL Eppendorf tube. The buffers were added to various concentrations of ammonium sulfate in 5 mM citrate buffer (pH 7.0). The final protein concentration was 50 μM. The protein was incubated in the test buffer at room temperature for 6 hours to achieve equilibrium. The sample was then centrifuged at 16,000 × g for 10 minutes. The concentration of the supernatant was measured using the Bradford assay with Coomassie Brilliant Blue G-250 dye and a Synergy H1 microplate reader (BioTek).

[0101] <Example 1> Identification of an ice-binding protein having ice-binding properties

[0102] <1-1> Identification of Ice-Binding Proteins

[0103] Experiments were conducted to identify ice-binding proteins with ice-binding properties.

[0104] Specifically, using the DELTA-BLAST (Domain Enhanced Lookup Time Accelerated BLAST) algorithm, 94 sequences with a sequence similarity of 40% or higher were identified using the LeIBP sequence (UniProt: C7F6X3) which lacks a signal sequence in BLASTp. To select ice-binding proteins with high solubility and stability, candidate proteins with an instability index (II) of less than 20 were selected using ProtParam, and candidate proteins with a melting temperature of 60 or higher were selected using the DeepSTABp program (Fig. 1a). Next, a model was generated using AlphaFold, and candidates with tyrosine or bulky residues in the ice-binding surface and loop region were excluded because they could cause steric hindrance during interaction with ice crystals, which could negatively affect high thermal hysteresis (TH) activity.

[0105] Consequently, among the 13 candidates, ice-binding proteins extracted from Candidatus Cryosericum odelliiSMC5 (WP_119120131.1 and WP_165849008.1) were selected (Fig. 1b), and WP_165846303.1 was additionally selected by analyzing the genome of C. odelliiSMC5 and named CoIBP1, CoIBP2, and CoIBP3, respectively (Fig. 1c).

[0106] <1-2> Analysis of the Domain Structure of Ice-Binding Proteins

[0107] The domains of CoIBP1, CoIBP2, and CoIBP3 were analyzed using InterPro.

[0108] As a result, as shown in Figure 1c, these proteins were found to contain multiple domains with secretion signal sequences at the N-terminus. All three proteins possess a DUF3494 ice-binding domain (IBD) (hereinafter CoIBD1, CoIBD2, and CoIBD3) and surface layer homology domains (SLHD) that interact with the cell wall via peptidoglycan, a bacterial lipopolysaccharide. While the IBD and SLHD are well conserved between proteins, each protein has unique characteristics. For example, CoIBP1 has a long linker consisting of 52 amino acids that is well conserved as a P-(X) repeat (X=Ala, Thr, Val, or Lys), whereas CoIBP3 has a short linker between the IBD and SLHD. Additionally, the inter-domain region of CoIBP2 contains a DUF11 domain similar to OmcB and an uncharacterized domain. The two domains are connected by five PX repeats.

[0109] The above results confirmed the identification of novel C. Cryosericum odellii-derived IBPs (i.e., CoIBP1, CoIBP2, CoIBP3) with multiple domains, along with DUF3494 IBD, which can be very stable and active, through bioinformatics analysis. The amino acid and base sequences of CoIBD1, CoIBD2, and CoIBD3 are shown in Tables 1 and 2 below.

[0110] 명칭서열(5'-3')서열번호CoIBD1MASASPAAVNLGTAGNFVILAKSGISTTGTTHVTGDIGVSPITATGMTGFGLTMDSSNTFATSALVTGKAYAADYTPPTPANMSTAVSDMETAYTAAAGVTAPAPVVELGAGNIGGMTLAPGVYKWSTGVTIPTDVTLAGGANDVWIFQIAQTLDLSNGIHVNLSGGAQAANIFWQVAGQTTLGTTSVFNGNILDQTAIVLNTGATLNGRALAQTAVTLDASTVSAS1CoIBD2MASPAPVDLGRAGDFVILAKSGISTSGATHVTGDIGVSPIDRTGLTGFSETMDPSNTFSTSTYVVAPGKLYAADYADPTPAKLTTAVSAMEAAYTDAGGRTGGLSVPGAGTILPATTLPAGVYTWSTGVTIPTGVTLEGGPDDVWIFQIAGTLDIATDMQVLLKGGAQAKNIFWQVGDVVTLHAGSHFEGNILGFSTIAMQTGASINGKLLSQKEVTLLGSDILTPAP2CoIBD3MAGPVAVVLGTAGNFVVLAKSGISTTGSTHVTGDIGVSPAAASAITGFGLTMDKSNTFSRSSLVTGKVYAADYHPPTPHNMTTAVSNMETAYTAAAGVTAPAPVVGLGAGNIGGMTIAPGVYKWSTGVTIPTNVTLAGGANDVWIFQIAQTLDISSAQKVILSGGAQAANIFWQVAGQTTLGTTSVFNGTILDLKAIVLNTGATLNGRALSQTAVTLDASTVSASVAA3

[0111]

[0112] <Example 2> Analysis of the activity of the ice-binding domain To verify the ice-binding characteristics of CoIBD, a recombinant protein was produced by cloning only the CoIBD portion from the entire sequence, and then experiments were performed to evaluate the thermal hysteresis (TH) activity and the ice-recrystalization inhibition (IRI) activity.

[0113] <2-1> Analysis of TH activity of the ice-binding domain

[0114] First, experiments were conducted to analyze the TH activity of CoIBD1, CoIBD2, and CoIBD3. AFPIII (Type III antifreeze protein), which exhibits moderate activity, was used as a control.

[0115] As a result, as shown in Figure 2, compared to the control group which showed TH activity of 0.3°C at 40 μM, CoIBD1, CoIBD2, and CoIBD3 showed TH activities of approximately 6.6°C, 4.1°C, and 3.1°C, respectively, at 40 μM. Generally, since the temperature hysteresis activity of ice-binding proteins is classified as high activity when it is 2.5°C, CoIBD1, CoIBD2, and CoIBD3 are ice-binding proteins with high activity.

[0116] During TH measurements, the control group AFPIII formed sharp double-pyramid ice crystals capable of growing single ice grains along the c-axis to form needle-shaped structures. CoIBD exhibited atypical morphology below the melting point. This was characterized by rupture into a dendritic pattern perpendicular to the c-axis at the non-equilibrium freezing temperature, and the initial shape of the ice crystal remained unchanged during rupture.

[0117] <2-2> Analysis of Ice Bonding Planes

[0118] To visualize the ice binding domain and the ice binding plane, a fusion protein of sfGFP (superfolder green fluorescent protein) and CoIBD was expressed and purified, and analyzed using fluorescence-based ice plane affinity (FIPA).

[0119] Specifically, the SfGFP-AFP III fusion protein was used as a positive control. The initial ice shape of the buffer containing CoIBD+GFP or SfGFP-AFPIII fusion proteins was evaluated to confirm that the fusion did not alter the binding plane of IBD.

[0120] As a result, as shown in Figure 2b, fluorescence signals were observed across the entire hemisphere of a single ice crystal hemisphere for three CoIBPs. Given that they covered the entire area of ​​the single ice crystal hemisphere, this suggests that CoIBDs coupled to multiple ice planes, including the basal plane. On the other hand, AFPIII coupled to the first-order prism and pyramid planes of the single ice crystal hemisphere. AFPIII coupled to the single ice crystal to form a hexagonal bipyramid shape.

[0121] Highly active ice-binding proteins generally have a TH activity range of 2–13°C, exhibit a dendritic pattern at freezing temperatures, and bind to multiple ice planes. Therefore, CoIBD1, CoIBD2, and CoIBD3 belong to a group of highly active ice-binding proteins capable of binding to all ice crystal planes. Since they bind to various ice crystal planes when constructing a purification system, it is expected that proteins can be bound to narrow ice surfaces with high yield, implying that they are ice-binding domains suitable for constructing a purification system.

[0122] <2-3> Analysis of IRI activity of the ice-binding domain

[0123] Next, to analyze the IRI activity of CoIBD1, CoIBD2, and CoIBD3, they were evaluated at various concentrations using the "splat cooling" method.

[0124] Specifically, a buffer or BSA-containing solution was dropped onto a cold aluminum block and then transferred to a low-temperature step set to -6°C to form several small ice granules. The growth of the ice granules was measured after 30 minutes of incubation.

[0125] As a result, as shown in Figures 3a and 3b, CoIBD inhibited ice growth, and the size of ice granules did not decrease despite an increase in IBD concentration. In contrast, ice recrystallization was observed in the absence of IBP or BSA, respectively. All CoIBDs effectively inhibited ice recrystallization even at 0.5 μM, and the ice size reached approximately 40% of that observed in the absence of the protein. The ice granule size did not change even with an increase in protein concentration. At 0.5 μM, CoIBD1 exhibited the highest IRI activity among the IBDs.

[0126] <Example 3> Preparation of a CoIBD1 variant (eCoIBD1) with improved thermal and chemical stability

[0127] <3-1> Thermal Stability and Sequence Analysis for Variant Design

[0128] Experiments were conducted to verify the thermal stability of CoIBD1, CoIBD2, and CoIBD3.

[0129] As a result, as shown in Figure 4a, the melting temperature (Tm) of CoIBD1 was the lowest at 62°C, while CoIBD2 and CoIBD3 showed high Tm values ​​of 71°C and 75°C, respectively.

[0130] CoIBD1 showed the highest TH activity among the three IBDs, but had low thermal stability. Therefore, the inventors designed a variant by comparing the sequence and structure of the IBD to produce a stable IBD.

[0131] Because CoIBD2 and CoIBD3 are more thermally and chemically stable, the inventors identified that residues conserved in the CoIBD2 and CoIBD3 sequences but not in CoIBD1 are involved in the hydrophobic core of the CoIBD1 structure (Figs. 4b and 4c). Additionally, efforts were made to maintain the high TH activity of CoIBD1 by avoiding interference with residues in the ice binding site and body region, which are indirectly involved in interactions with ice. Sequence and structural analysis revealed three residues (Met74, Ala88, Ala97) in the capping head region. These were substituted with residues from CoIBD3 (M74I, A88S, A97V) (Fig. 4d). The amino acid and base sequences of the designed CoIBD1 variant (eCoIBD1) are shown in Tables 3 and 4 below.

[0132] Name Sequence (5'-3') Sequence Number eCoIBD1MASASPAAVNLGTAGNFVILAKSGISTTGTTHVTGDIGVSPITATGITGFGLTMDSSNTFATSALVTGKVYAADYTPPTPANMSTAVSDMETAYTAAAGVTAPAPVVELGAGNIGGMTLAPGVYKWST GVTIPTDVTLAGGANDVWIFQIAQTLDLSNGIHVNLSGGAQAANIFWQVAGQTTLGTTSVFNGNILDQTAIVLNTGATLNGRALAQTAVTLDASTVSASAAAGGSGGDDDDKASMTGGQQMGRGGSEFELRRQACGRTRAPPPPPLRSGC4

[0133] 명칭서열(5'-3')서열번호eCoIBD1atggctagcgcaagcccggcagcagttaatctgggtacggcaggcaattttgttattttggcaaaatcaggtatcagcacaacaggtactacacatgttacaggtgacataggtgtttctccgattaccgcaacaggcatcaccggatttggtcttaccatggattcttcaaatacctttgcaacctctgcattagttaccggtaaagtatatgcagcagactacactccaccaactccagcaaatatgtcgacagccgtaagcgatatggagactgcatataccgcagcagcaggtgttaccgcaccggcaccggtcgttgaattaggtgcaggtaacattggtggtatgactctggcacccggtgtttataagtggagcacgggtgttaccattcctactgatgtaaccctggcaggaggtgcaaatgacgtttggattttccagattgcacaaaccctggatctttcaaatggtatacatgttaatttgagcggtggtgcacaggcagcaaacattttctggcaggtagcaggtcaaacgaccctcggtaccacaagcgttttcaatggtaatatactggatcagacggcaattgttctcaataccggtgcaacgttaaatggtcgtgcattggcgcaaaccgcagtaactttagatgcaagcaccgttagtgcttcggcagcagcaggtggttctggtggtgacgacgacgacaaggctagcatgactggtggacagcaaatgggtcgcggatccgaattcgagctccgtcgacaagcttgcggccgcactcgagcaccaccaccaccaccactgagatccggctgctaa8

[0134] <3-2> 변이체의 열 안정성 분석CoIBD1의 잔기가 치환된 돌연변이체의 열 안정성을 분석하였다.

[0135] As a result, as shown in Figure 4e, the Tm of M74I and A97V increased by 0.3°C and 3.3°C, respectively, while A88S decreased by 1°C compared to wild-type CoIBD1 (61.4°C). The double mutants M74I and A97V showed greater stability than the single mutants (M74I or A97V), with an average Tm of 68.5°C. A structural comparison of wild-type CoIBD1 and the double mutants (M74I / A97V) revealed that mutations in each residue increased hydrophobic interactions with surrounding residues. However, single or double combination mutants with A88S did not show increased stability, indicating that the hydrophobic core of the capping head region of IBD is important for protein stability. Additionally, the double mutants (M74I / A97V, named eCoIBD1) maintained TH and IRI activities similar to wild-type CoIBD1. Therefore, a thermally stable and chemically stable IBD (eCoIBD1) was designed by mutating two residues in the capping head region without interfering with the high activity of CoIBD1 in a recombinant protein purification system using ice-binding proteins.

[0136] <Example 4> Purification of recombinant protein using an ice-binding domain

[0137] <4-1> Construction of a Purification System Using Ice-Binding Domains and Analysis of Purity and Efficiency

[0138] To construct a purification system using an ice-binding domain, an expression vector containing the eCoIBD1 ice-binding domain gene designed from Example 3 above and a Multi Cloning Site (MCS) was constructed. This vector was constructed based on the pET28a vector and included an Enterokinase restriction enzyme site and a linker between eCoIBD1 and the MCS. This is to remove the ice-binding domain after purifying the target protein.

[0139] For a protein purification system using an ice-binding domain, a device combining a water chiller circulator and a bottle rotation system was used. A bottle filled with dry ice was rotated for about 10 minutes to create ice inside the bottle, and the thickness of the ice was about 2–3 mm (Fig. 5a).

[0140] The first purification showed a purity of 55% for the ice fraction, and subsequent purification using the ice fraction from the first purification resulted in a purity of 87% for the target protein (Figs. 5b and 5c). Efficiency calculations showed that 29% of the target protein was recovered after two purifications (Fig. 5c).

[0141] <4-2> Analysis of Ice Binding Characteristics of the Ice Binding Domain

[0142] The ice was cut vertically to confirm the distribution of IBD bound to the ice bottom through fluorescence analysis.

[0143] As a result, as shown in Figure 6a, it was confirmed that the ice was separated into the ice bottom and the IBD bonding portion.

[0144] In addition, as a result of observation with a confocal microscope, it was confirmed that the fluorescence of the part bound to the IBD was evenly distributed, as shown in Fig. 6b.

[0145] In addition, when observing the ice from above, as shown in Figure 6c, it was found that IBD-GFP could bind to the ice regardless of depth.

[0146] These results indicate that the eCoIBD-GFP fusion protein can extensively bind to ice surfaces, as observed in the FIPA analysis.

[0147] <Example 5> Effect of an additive in the buffer of a purification system using an ice-binding domain

[0148] To determine the effect on a protein purification system using an ice-binding domain, an experiment was conducted including an additive during protein purification.

[0149] Protein purification buffers contain various additives to prevent protein damage. These additives include the reducing agent DTT (dithiothreitol), the protein degradation inhibitors PMSF (phenylmethanesulfonyl fluoride), EDTA (ethylenediaminetetraacetic acid), and AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride), the osmotic agents glycerol and Triton X-100, and sugar. Accordingly, the effects of including these additives in the buffer during protein purification on a protein purification system utilizing an ice-binding domain were investigated.

[0150] Specifically, glycerol (5%), 1 mM EDTA, or 1 mM DTT was added to the buffer during purification.

[0151] As a result, as shown in Figure 7, it was confirmed that the addition of specific buffer additives (glycerol, EDTA, EDTA) during protein purification did not affect the yield or purity of the purification system. In particular, the yield and purity of 5% glycerol were 32% and 88%, respectively. In the case of 1 mM DTT, the yield was 39% and the purity was 78%. Similarly, when using 1 mM EDTA, the yield was 34% and the purity was 91%.

[0152] These results suggest that additives can be applied to protein purification systems using ice-binding domains.

[0153] <Example 6> Analysis of Solubility and Expression Levels of Ice-Binding Domains

[0154] An experiment was conducted to verify the solubility of eCoIBD1.

[0155] As a result, as shown in 8a, this protein maintained solubility even at high concentrations of ammonium sulfate.

[0156] In addition, experiments were conducted to confirm the expression level of eCoIBD1 in E. coli.

[0157] As a result, as shown in 8b, it was confirmed that the expression level of eCoIBD1 in E. coli was significantly high.

[0158] These results suggest that the high solubility of the ice-binding domain and the enhanced expression level of the target protein can be usefully employed as a protein purification system.

[0159] In conclusion, the present invention relates to a novel protein purification system using an ice-binding domain. The ice-binding domain of the present invention has the characteristic of binding to various crystal lattice planes and has high activity, high solubility, and high expression levels, so it can be usefully used to purify a target protein by utilizing these characteristics (Fig. 9).

Claims

1. An ice-binding domain comprising an amino acid sequence selected from the group consisting of sequence numbers 1 to 4.

2. Comprising the ice-binding domain of claim 1 and the target protein, Fusion protein.

3. In Paragraph 2, The above ice-binding domain is characterized by being encoded by a nucleotide sequence consisting of SEQ ID NOs 5 to 8. Fusion protein.

4. In Paragraph 2, The above target protein is characterized by being connected to the N-terminus or C-terminus of the ice-binding domain. Fusion protein.

5. In Paragraph 2, Characterized by additionally including a protein restriction enzyme cleavage site between the ice-binding domain and the target protein. Fusion protein.

6. A polynucleotide encoding the fusion protein of paragraph 2.

7. An expression vector comprising the polynucleotide of claim 6.

8. A host cell transformed with the expression vector of claim 7.

9. (1) A step of preparing a solution comprising a fusion protein comprising the ice-binding domain of claim 1 and the target protein; (2) a step of inducing adsorption of the recombinant protein by placing the above solution into a container containing ice; and (3) A step of separating the target protein from the fusion protein, Method for purifying recombinant proteins.

10. In Paragraph 9, The above (2) step is characterized by being performed under conditions of 0℃ or lower, method.

11. In Paragraph 9, The above step (2) is characterized by being performed by a water chiller circulator, method.

12. In Paragraph 9, The above (3) step is characterized by being performed by treatment with a protein cutting enzyme, method.

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