KR101581576B1

KR101581576B1 - Method for The Preparation of Amylose Magnetic Bead and Its Application for Protein Purification

Info

Publication number: KR101581576B1
Application number: KR1020140115442A
Authority: KR
Inventors: 김영록; 임민철; 이관형
Original assignee: 경희대학교 산학협력단
Priority date: 2014-09-01
Filing date: 2014-09-01
Publication date: 2015-12-31

Abstract

A method for producing amylose magnetic beads and a method for purifying proteins using the same are disclosed.
The method of purifying a target protein using amylose magnetic beads according to the present invention comprises the steps of (a) preparing amylose synthase and one or more substrate selected from glucose, oligosaccharide, and sucrose, and a guest molecule ) To produce an amylose magnetic bead complex; (b) binding the target protein to the amylose magnetic bead complex; And (c) separating and purifying the amylose magnetic bead conjugate to which the target protein is bound.

Description

TECHNICAL FIELD The present invention relates to a method for preparing amylose magnetic beads and a method for purifying proteins using the same,

The present invention relates to a method for producing amylose magnetic beads and a method for purifying proteins using the same, and more particularly, to a method for preparing an amylose magnetic bead conjugate and a method for separating and purifying a protein having maltose binding protein (MBP) .

A variety of ligands have been attracting attention in recent decades for applications in the separation and purification of proteins, which can bind proteins with high affinity and selectivity (Young et al. of affinity tags and microbial applications, Ha et al 2008 Purification of his-tagged proteins using Ni2 + -poly (2-acetamidoacrylic acid) hydrogel). Selective separation and purification of proteins using complex matrices is an important technology for protein and enzyme engineering. The use of affinity tags using recombinant DNA technology allows easy expression and purification of recombinant proteins. In addition, intimacy tags are useful tools for increasing protein expression efficiency, characterizing protein structures, and increasing protein solubility. One of the affinity tags, maltose binding protein (MBP), is a 42 kDa protein encoded by the mal E gene of E. coli K12 (Duplay et al 1988). Two regions of mature periplasmic maltose-binding protein of Escherichia coli involved in secretion. The maltose binding protein increases the solubility of the expressed protein and has resistance to protein degradation, and the affinity of the maltose binding protein for amylose has been widely used for protein expression and purification since it can simplify protein purification.

For purification of proteins, columns containing resins that are typically functionalized with a specific ligand have been used, and these methods are suitable for large-scale protein purification and mild elution conditions (Gutierrez et al 2007 Immobilized metal-ion affinity chromatography: trends). However, this technique has the disadvantage that the purification time is long due to slow diffusion and pressure drop in the resin (Zou et al 2001 Affinity membrane chromatography for the analysis and purification of proteins). In order to overcome the disadvantages of column-based affinity chromatography, other types of separation systems have been developed. In order to overcome the problems of resin-packed particle chromatography, a macroporous and microporous synthetic membrane was used (Ghosh 2002 Protein separation using membrane chromatography: opportunities and challenges). Microfluidic systems filled with functional microfibers, which are chip-based protein separation systems, have also been developed (Jo et al 2010 Microfluidic channels fabricated on mesoporous electrospun fiber mats: A facile route to microfluidic chips). Recently, there has been an increasing interest in magnetic bead-based magnetic separation techniques that do not require bulky liquid chromatography systems, centrifugation, filtration or other devices (Safraik et al 2004) ).

In order to improve dispersion, biocompatibility and further functionality, the surface of the magnetic beads can be easily functionalized with a capping material, an inorganic metal, a surfactant, and a polymer. However, little research has been done on the production of magnetic beads for the isolation and purification of MBP-fusion proteins (Zhou et al., 2012). Maltodextrin-binding protein (MBP) fusion enzymes, modified magnetic microspheres for selective enrichment of maltose binding proteins. For example, for the production of magnetic beads coated with a ligand such as maltose and maltodextrin, functionalization of the surface requires complex processing such as pretreatment, cleaning and cross-linking chemical treatment. In addition, the ability to purify magnetic beads is highly dependent on the grafting density of the ligand functionalized primarily on the magnetic bead surface by chemical bonding.

Thus, the present inventors have formed an amylose magnetic microbead complex by capturing magnetic nanoparticles by an enzymatic synthesis process using amylose. The present inventors have completed the present invention by confirming that the amylose molecule on the surface of the amylose magnetic microbeads can be used for affinity adsorption with the MBP-fusion protein.

Korean Patent Registration No. 1214572 (published on Mar. 27, 2012)

It is an object of the present invention to provide a method for purifying MBP-tagged proteins by preparing amylose magnetic beads.

In order to accomplish the above object, the present invention provides a method of manufacturing amylose magnetic beads, wherein the amylose magnetic bead according to an embodiment of the present invention comprises amylose synthase, glucose, oligosaccharide, sucrose, and a magnetic nanoparticle as a guest molecule to produce an amylose magnetic bead complex.

The present invention also provides a method for purifying a target protein using amylose magnetic beads. The method for purifying a target protein using amylose magnetic beads according to an embodiment of the present invention comprises the steps of: (a) mixing amylose synthase with glucose, preparing at least one amylose magnetic bead complex by reacting at least one substrate selected from the group consisting of oligosaccharide and sucrose and a magnetic nanoparticle as a guest molecule; (b) binding the target protein to the amylose magnetic bead complex; And (c) separating and purifying the amylose magnetic bead conjugate to which the target protein is bound.

Hereinafter, the present invention will be described in more detail.

In the method for producing the amylose magnetic beads of the present invention and the method for purifying the target protein using the amylose magnetic beads, the amylose synthase may be selected from the group consisting of amylosucrase, phosphorylase, starch synthase ), Amylase, and D-enzyme (D-enzyme) are preferably used, and amylosucrase is more preferable.

Further, in the method for producing the amylose magnetic bead of the present invention and the method for purifying the target protein using the amylose magnetic beads, the magnetic nanoparticles are preferably iron oxide nanoparticles, and the target protein is maltose binding proteins (MBP) Protein (MBP-tagged protein).

In addition, in the method of purifying a target protein using the amylose magnetic bead of the present invention, the step (c) includes a step of adding a competitive binder to the target protein on the surface of amylose to separate the target protein from the amylose magnetic bead .

Hereinafter, the present inventors demonstrate their application to the rapid and efficient synthesis of amylose magnetic beads composed of pure amylose molecules and iron oxide nanoparticles through amylosechocase-mediated catalysis and magnetic separation and purification of MBP-fusion proteins . The use of an enzyme catalyst based on a bottom-up approach allows the iron oxide nanoparticles to react with the substrate to form an amylose magnetic bead complex. Amylose molecules on the surface of amylose magnetic beads can be used for affinity adsorption with MBP-fusion proteins. The efficiency of amylose magnetic beads as a template for magnetic separation and purification of proteins could be demonstrated by MBP-fusion green fluorescent protein (GFP) as a model protein. In order to study the application in biomolecule separation, the selectivity and recycling of amylose magnetic beads were investigated. Amylose magnetic beads can be recycled several times without loss of efficacy in the purification capacity of the target protein.

Specifically, the enzymatic synthesis of amylose microparticles in vitro is a powerful bottom-up approach for the production of amylose-nanomaterial hybrid microparticles.

Hereinafter, the present inventors report a method for the enzymatic synthesis of amylose using amylocyte scraping and the production of amylose magnetic beads by the introduction of iron oxide nanoparticles as a guest molecule. Iron oxide nanoparticles can form complexes with amylose molecules synthesized by hydrophobic reaction of amylose and iron oxide nanoparticles during an enzymatic reaction. Because of their magnetic properties, amylose magnetic microparticles make it possible to use them for magnetic separation applications of biomaterials. The magnetic separation force was measured by testing separation and purification of MBP-fused GFP from E. coli cell solution using amylose magnetic beads, and the purification capacity was 72 μg of amylose magnetic bead mg protein. Thus, the amylose magnetic beads can be used to isolate the MBP-fusion protein and detect the target molecule using the amylose magnetic bead functionalized with the ligand-fused MBP protein.

The amylose magnetic beads according to the present invention can be used for separating MBP-fusion proteins and detecting target molecules using amylose magnetic beads functionalized with a ligand-fused MBP protein. The present invention also provides a simple way to manufacture amylose magnetic beads that can be applied in various fields such as biosensors and actuators in the near future for small volume protein separation and purification.

Figure 1 is a schematic diagram illustrating the enzymatic synthesis of amylose-nanoparticle microparticles (AM-NP microparticles) prepared according to an embodiment of the present invention.
FIG. 2A is a scanning electron microscope image (SEM) (size bar is 2 μm) observing the amylose magnetic beads produced according to an embodiment of the present invention, FIG. 2B is a scanning electron microscope image (SEM) (A) reaction without iron oxide nanoparticles, (b) reaction with iron oxide, and (c) reaction without amylocyte crusher).
FIG. 3 is a graph showing the results of XRD analysis of amylose beads produced by the examples of the present invention, wherein (a) is iron oxide nanoparticles, (b) amylose magnetic beads, and (c) amylose beads.
FIG. 4A shows magnetic hysteresis loops of magnetic beads of (a) amylose beads and (b) amylose magnetic beads prepared according to an embodiment of the present invention, and (B) In order to show that the micromagnetic amylose beads synthesized by the example can be selectively separated from the substrate solution by the magnet for 30 seconds, a comparison between the state where the magnet is not approaching the substrate solution and the state where the magnet is approaching the substrate solution This is the picture shown.
5 (A) and (B) are photographs showing separation of (A) His-fused GFP and (B) MBP-fused GFP in the cuvette after 30 minutes of reaction with the amylose magnetic beads prepared according to the embodiment of the present invention, (C) and (D) show the results of (C) His-fused GFP and amylose magnetic beads and (D) MBP-fused GFP after washing the amylose magnetic beads three times with the magnet in the reaction solutions (A and B) A fluorescent microscope image of amylose magnetic beads (at this time, the size rod is 2 μm).
6 is an SDS-PAGE analysis (M, protein size marker, CL, fraction of cell lysate, W; FIG. 6) showing the result of purification of MBP-fused GFP of amylose magnetic beads prepared according to the example of the present invention. Washed, and E: eluted fraction).
7 shows the results of analysis of the recyclability of amylose magnetic beads prepared according to the example of the present invention for MBP-fusion GFP purification (A) through Bradford analysis and (B) SDS-PAGE analysis ; Protein size marker, CL; fraction of cell lysate, 1-3; MBP-fusion GFP released from amylose magnetic beads reused three times).
FIG. 8 shows the structure of the pMal-c2x :: histag / gfp vector of the present invention. The Histag- gfp fragment having the EcoRI and XbaI enzyme resistance sequences was ligated with pMal-c2x using T4 DNA ligase.
9 is an electrophoresis image showing the expression of MBP-His-GFP protein in the pMal-c2x :: histag / gfp vector ((A) Amylose resin and (B) Purification of MBP-His- Ni-NTA resin was used (M: marker, P: pellet, F: flow, W: washing solution, and E;
10 is a TEM image, electron mapping, and EDX spectrum of the amylose magnetic beads of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. However, it should be understood that the following embodiments are provided so that those skilled in the art may understand the present invention without departing from the scope and spirit of the present invention. It is not.

&Lt; Example 1 > Chemicals and bacterial strains

Sucrose, maltose, Tris-HCl (tris (hydroxymethyl ) aminomethane hydrochloride), NaCl, NaH 2 PO 4, SDS, published imidazole, Amphitheater, IPTG and magnetic iron oxides nanoparticles Sigma-Aldrich (St. Louis, MO, USA) Lt; / RTI > LB (Luria-Bertani) solution was purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). All experiments were conducted using distilled water. Escherichia coli MC1061 was used as a host for a pHCE vector (pHCDGAS) encoding recombinant DGAS. And E. coli DH5? Containing pMal-c2x :: histag / gfp was used for overexpression of MBP-fusion protein

<Example 2> Synthesis of amylose magnetic beads

DGAS ( Deaminococcus geothermalis- derived amylocyte clase) was prepared by the method previously used by the present inventors. In summary, recombinant E. coli MC1061 harboring pHCDGAS was grown in 500 ml LB culture (0.1 mg / ml ampicillin) at 250 rpm at 37 C for 24 hours. The cells were harvested with centrifugation (7,000 x g for 20 minutes at 4 ° C) and washed with lysis buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, and 250 mM imidazole (pH 8.0)) . The activity of the purified enzyme was determined by preparing a standard curve using fructose and determining the hydrolytic activity by the dinitrosalicylic acid (DNS) method (Sumner et al, A method for determination of saccharase activity J. Biol. Chem. 1935, 108, 51-54). To prepare the amylose beads, an aqueous solution containing 500 mM sucrose, 300 U DGAS in 1 ml 50 mM Tris-HCl buffer (pH 7.0) was prepared and the enzyme reaction was routinely performed at 30 ° C for 24 hours. Amylose magnetic beads were prepared by adding 200 μl of heptane containing iron oxide nanoparticles (<14 mg / ml) to 1 ml of the aqueous reaction solution. Prior to the addition of DGAS, the reaction solution was mixed by bath sonication for 5 minutes to disperse the organic phase in the aqueous solution. Thereafter, 300 U DGAS was added to the mixed reaction solution and the reaction was performed under the same conditions as described above. After completion of the enzymatic amylose synthesis reaction, the amylose beads and the amylose magnetic beads were harvested by centrifugation and magnets, respectively. The collected particles were washed several times with purified water and stored in 20% ethanol at 4 ° C until further use.

Amylose magnetic beads were prepared by amylocosche catalyst synthesis based on a bottom-up approach that provides a novel way to complex with guest molecules such as iron oxide nanoparticles. Enzymatic polymerization of amylose molecules began by hydrolyzing sucrose to fructose and glucose. Later, amylose cyclase used glucose molecules as the first receptor for the amylose polymerization process. By repeating the enzymatic reaction cycle, malto-oligosaccharides and longer amylose chains were prepared and the amylose chains were self-assembled by hydrophobic interaction and hydrogen bonding (Fig. 1). During the enzymatic synthesis of the amylose molecule, the guest molecule can readily complex with the amylose molecule. In the present invention, hydrophobic iron oxide nanoparticles are used as guest molecules to enable amylose beads to have magnetic characteristics. However, phase separation occurs due to the property that the water used as the solvent for the amylose cyclase reaction and the heptane in which the iron oxide nanoparticles are dispersed are not mixed with each other. Prior to the addition of the enzyme in the reaction solution, the dispersion of the organic solvent was induced by using ultrasonic waves to increase the interface between the solutions and the dispersion was maintained by an angle adjustable rotator.

&Lt; Example 3 > Structural and morphological characteristics of amylose magnetic beads

<3-1> Scanning Electron Microscope (SEM)

The prepared amylose magnetic bead solution was dropped onto the plasma-treated silicon substrate and dried at ambient temperature. The morphology of the amylose magnetic beads was confirmed using a field emission scanning electron microscope (FE-SEM; Leo Supra 55, Genesis 2000, Carl Zeiss, Oberkochen, Germany) with an acceleration voltage of 5 kV. The average size of the prepared amylose magnetic beads was determined by measuring 100 particle diameters from the SEM image.

Enzymatically synthesized amylose molecules were bound together in solution and formed into microsized spherical particles in vitro. The prepared amylose magnetic beads have a spherical shape (Fig. 2A). The average size of the amylose magnetic beads was determined by measuring the diameter of the particles from an SEM image, which is one of the commonly used methods for determining the mean size of fine particles or nanoparticles. Through analysis, the average diameter of the amylose magnetic beads produced was 2.09 ± 0.42 μm. After completion of the enzymatic reaction, a brown precipitate was observed (Figure 2B-b) due to complex formation with iron oxide nanoparticles, whereas a white precipitate was observed in the control solution without iron oxide nanoparticles (Figure 2B-a ). In the absence of DGAS, iron oxide nanoparticles aggregated on the inner surface of the reaction tube after the organic solvent had completely evaporated (FIG. 2B-c). This means that the amylose molecules synthesized during organic solvent evaporation are complexed with iron oxide nanoparticles and that the nanoparticles are stabilized in an aqueous solution. During the enzymatic amylose synthesis reaction, gradual evaporation of the organic solvent enables complex formation between the synthesized amylose molecule and iron oxide nanoparticles.

&Lt; 3-2 > X-ray diffraction (XRD)

Ray diffraction pattern of three samples (iron oxide nanoparticles, amylose beads, and amylose magnetic beads) from 10 to 80 degrees (2?) Using Cu K? Radiation in a Bruker D8 Advance diffractometer (Bruker, Karlsruhe, Germany) Respectively. For the preparation of XRD samples, droplets of iron oxide nanoparticles were dropped onto a glass cover and dried. Amylose beads and amylose magnetic beads were dehydrated in a vacuum dryer and analyzed by an X-ray diffractometer.

Figure 3 shows X-ray diffraction (XRD) of iron oxide nanoparticles, amylose beads, and amylose magnetic beads. From the XRD pattern, amylose beads (curve c) show diffraction peaks at 2 ? = 15, 17, 22, and 24 degrees derived from the pure B-form crystal structure of amylose (Potocki-Veronese et al 2005 Amylose synthesized in vitro by amylosucrase: morphology, structure, and properties). These results indicate that the particles produced by DGAS can be used for the separation and purification of MBP-fusion proteins. The iron oxide nanoparticles (curve a) exhibit typical Fe ₃ O ₄ patterns in which the refraction peaks are observed at 2 θ = 30, 35, 43, 57, and 62 ° (Li et al 2012 Magnetite-loaded fluorine-containing polymeric micelles for magnetic resonance imaging and drug delivery). In addition, the amylose magnetic beads (curve b) have all peaks for the amylose beads and iron oxide nanoparticles produced. The sharpness and intensity of the amylose peak of curve b were reduced compared to curve c. This means that the crystallinity of amylose was slightly destroyed by the inserted iron oxide nanoparticles. However, the degree of crystallinity does not lead to a large change in the affinity for the MBP of the formed amylose magnetic beads. The XRD data shows that the produced amylose magnetic beads are composed of pure amylose molecules and iron oxide nanoparticles.

<3-3> Vibrating sample magnetization (VSM)

A vibrating sample magnetometer (VSM) (LakeShore 7404, Lake Shore Cryotronics, Inc., Westerville, OH, USA) was used to verify the magnetic properties of the amylose beads and amylose magnetic beads. A sample solution of amylose beads and amylose magnetic beads was prepared at the same mass concentration per volume.

The magnetic properties of the amylose beads and amylose beads were analyzed by a VSM (vibrating sample magnetization) magnetometer. The hysteresis loop of amylose beads (a) and amylose magnetic beads (b) is shown in Figure 4A. There is a remarkable difference between the two samples. Although amylose beads alone do not have any magnetic properties, amylose magnetic beads have superparamagnetic properties. The saturation magnetization value of the prepared amylose magnetic beads is 4.5 emu / g. Amylose magnetic beads can be dispersed in water by pipetting or shaking and exhibit a brown suspension. The amylose magnetic beads were easily separated from the dispersion by an external magnetic field within 30 seconds. Further, the separated amylose magnetic beads were redispersed in the solution by magnetic field removal and shaking (FIG. 4B). These results indicate that the prepared amylose magnetic beads have magnetic ability and redispersibility for practical application such as magnetic separation.

Example 3 Expression and Purification of MBP-Fusion Protein from Cell Lysate

E. coli DH5? Containing pMal-c2x :: histag / gfp was cultured for overexpression of MBP-fusion green fluorescent protein (GFP). 100 ml were cultured at 37 占 폚 with constant stirring in LB medium containing ampicillin (100 占 퐂 / ml). After reaching OD ₆₀₀ = 0.8, the culture was over-induced with 0.1 mM IPTG at 18 ° C overnight. The cells were then pelleted by centrifugation at 3,000 x g for 20 minutes at 4 ° C and redispersed in column buffer (20 mM Tris-HCl, 200 mM NaCl, and 1 mM ETDA, pH 8.0). Cell destruction was performed using an ultrasonic wave (VC 750, Sonics & Materials Inc., Newtown, CT, USA) for 15 min in an ice bath for 10 min and at 25 sec intervals. A soluble fraction of the cell disruption was obtained by centrifugation at 3,000 x g for 20 minutes at 4 占 폚. 1 ml of the soluble fraction was reacted with 5 mg of amylose magnetic beads at 4 ° C for 30 minutes using a tube rotator (AG, FinePCR, Seoul, Korea). The amylose magnetic beads were then separated by magnets and washed 3 times with 1 ml of column buffer to remove unbound lysates. Subsequently, 200 mu l of elution buffer (column buffer + 10 mM maltose) was added and reacted at 4 DEG C for 10 minutes to elute MBP-GFP. All fractions of each step were analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the amount of purified MBP-GFP was determined by Bradford analysis. To confirm the recyclability of amylose magnetic beads, the same procedure as described above was used to study the recyclability of amylose magnetic beads for purification of MBP-fusion proteins. After each final elution, the amylose magnetic beads were washed in the following order: 300 μl of water, 300 μl of 0.1% SDS, 100 μl of water and 500 μl of column buffer. Recycling was tested 3 times and the eluted fractions were analyzed by SDS-PAGE and Bradford measurements.

In order to demonstrate the utility of amylose magnetic beads in the isolation and purification of MBP-fusion proteins from cell lysates, amylose magnetic beads were reacted with a cell lysate containing overexpressed MBP-fused GFP and separated by magnets. First, the selectivity of the amylose magnetic beads prepared above to the MBP-fusion protein was examined by testing the binding and separation of the MBP-fusion protein to the amylose magnetic beads. GFP with different fusion of His- and MBP- was isolated and mixed with amylose magnetic beads at the same concentration. Figures 5A and 5B show photographs of proteins separated by affinity reaction and magnetic field. In MBP-fused GFP, the color of the solution changed from green to colorless, and greens remained green even after the accumulation of magnetic particles in His-fused GFP. The collected amylose magnetic beads were washed several times with PBS buffer and observed with a fluorescence microscope. Only strong green fluorescence was released in MBP-fused GFP and amylose magnetic beads (Fig. 5D), whereas no green emission was observed in His-fused GFP (Fig. 5C). This indicates that the MBP-fusion protein is selectively attached to the surface of the amylose magnetic beads.

Purification of the MBP-fusion protein of the prepared amylose magnetic beads was carried out using MBP-fusion GFP. From cell lysates, MBP-fusion proteins were selectively attached to amylose magnetic beads and separated by magnetic fields. By introducing an elution buffer containing 10 mM maltose, a competitive binder on the amylose surface, the captured MBP-fused GFP protein was released from the amylose magnetic beads. As shown in Figure 6, all fractions of cell lysate, wash liquor and eluate were analyzed by SDS-PAGE. These results suggest that amylose magnetic beads are specific for MBP-fusion proteins and pure GFP (72 kDa) can be obtained by the introduction of a maltose solution (10 mM). The amount of eluted protein was also measured by Bradford analysis. The purification capacity of amylose magnetic beads for MBP-fused GFP was determined to be 72.31 g per mg of amylose magnetic beads. The purification capacity of the amylose magnetic beads for the MBP-fusion proteins of the present invention is significantly greater than the capacity of commercial NEB amylose magnetic beads (19.75 ug / magnetic bead mg) (Table 1).

Comparison of amylose magnetic beads synthesized in the Examples and MBP-fusion protein purification capacity of commercial products matter Tablet capacity Test protein Example 72.31 [mu] g / mg MBP-GFP (72 kDa) NEP (E8035S) 19.75 [mu] g / mg MBP-GFP (72 kDa)

In addition, the tablet capacity of the present invention is also greater than the capacity of maltodextrin-modified magnetic microspheres (22 μg / magnetic bead mg) produced by chemical conjugation (Zheng et al 2014 Maltodextrin-modified magnetic microspheres for selective enrichment of maltose binding proteins. The difference in purification capacity of the magnetic beads produced by the enzyme-biocatalytic bottom-up appraoch of the present invention and the magnetic beads produced by the chemical bond-based approach is due to the surface coverage of the specific ligand ). Because magnetic beads for MBP-fusion protein binding are typically prepared with covalent linkages of specific ligands, such as maltodextrin and maltose, on the surface of paramagnetic beads (Zheng et al. 2014, Maltodextrin-modified magnetic microspheres for selective enrichment of maltose binding proteins, Zhou et al. For this reason, the grafting density of the specific ligands on the surface of the magnetic beads is considered to be a major factor in the chemical linkage based method. However, the amylose magnetic beads prepared in the present invention are composed of an enzyme-synthesized amylose molecule acting as a matrix material for complex formation with iron oxide nanoparticles and also as an affinity ligand for MBP-fusion protein separation. In this approach, the inventors have been able to eliminate the complicated process for linking specific ligands to magnetic bead surfaces and to improve the purification capacity of MBP-fusion proteins.

In addition, the recyclability of amylose magnetic beads was tested using MBP-fused GFP as the target protein. After each final protein elution, excess maltose was removed and the surface of the bead was cleaned by washing the magnetic beads with 300 μl of water, 300 μl of 0.1% SDS, 100 μl of water and 500 μl of column buffer. The relative purification capacity of the amylose magnetic beads for the MBP-fusion protein during 3 re-use and the SDS-PAGE analysis results are shown in FIG. From Bradford analysis, the tablet capacity was reduced to 93% at the second use and to 88% at the third use (Fig. 7A). SDS-PAGE analysis also showed that MBP-fused GFP was efficiently purified by amylose-source magnetic beads (Fig. 7B). However, the reduction in tablet capacity can be attributed primarily to the loss of amylose magnetic beads during sample handling such as pipetting. That is, amylose magnetic beads maintain affinity and specificity for the MBP-fusion protein after three regeneration and reuse. Therefore, the amylose magnetic beads prepared in the present invention are suitable for separation and purification of MBP-fusion protein from cell lysate.

Example 4: Design and purification of MBP-His-GFP

<4-1> Chemicals

Agar powder, NaCl (sodium chloride) and sodium hydroxide (NaOH) were purchased from Daejung Chemicals & Metals Co., Ltd. (Kyonggi-do, Korea). LB (Luria-Bertani) solution was purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). The electron-cuvette (0.2 cm) is from Bio-Rad Company (Hercules, Calif., USA). Ampicillin was purchased from Biosesang company (Kyonggi-do, Korea). Other chemicals such as T4 DNA ligase, EcoRI, XbaI, and amylose resin were purchased from New England Biolabs (Ipswich, Mass., USA). Ni-NTA resin was purchased from Qiagen (Valencia, CA, USA). NaH ₂ PO ₄ , imidazole, and lysozyme (egg whites) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PCR SV kit for PCR product purification was purchased from GeneAll (Seoul, Korea).

<4-2> Design of MBP-His-GFP

In order to produce the MBP-His-GFP expression vector, pET28a :: GFP vector of the histag sequence 18-bp, EcoRI resistance 6-bp and 6-bp projecting a forward primer (5 'GCA TCA GAA TTC CAT CAT containing the enzyme (5 ' TAC AGT TCT AGA ( CAT CAT CAT CAC 3' ) and 18-bp corresponding to the gfp gene in the pET28a :: GFP vector with XbaI resistance enzyme 6-bp, termination codon sequence and overhang 6-bp The histag-gfp fragment was amplified from pET28a :: GFP vector by PCR using TTA TTT GTA TAG TTC ATC CAT3 '. The amplified histag-gfp product was purified and digested with EcoRI and XbaI resistance enzymes. The vector skeleton was prepared by digesting the pMal-c2x vector with these two enzymes. Linkage of these vectors to the enzyme digested PCR products was performed using T4 ligase according to the manufacturer's instructions and the mixture was transformed into E. coli DH5a by electroporation (Figure 8). Scanning for positive colonies was tested by colony PCR using the primers.

<4-2> Purification of MBP-His-GFP

E. coli DH5? PMal-c2x :: histag / gfp were cultured overnight in 100 ml LB with 100 占 퐂 / ml ampicillin at 37 占 폚 and 200 rpm. When the culture reached an optical density of about ~ 0.8 (at 600 nm) or more, 0.1 mM IPTG was added to express the MBP-His-GFP protein. Overexpression was induced at 220 rpm, 18 < 0 > C overnight. The cells were then harvested by centrifugation at 3,000 x g for 20 min at 4 < 0 > C and were divided into two groups. Groups 1 and 2 were suspended in the appropriate buffer solution, sonicated, and impurities were removed by centrifugation at 3,000 x g for 20 minutes at 4 ° C. Group 1 was suspended in Digestion Buffer _{(50 mM NaH 2 PO 4,} 300 mM NaCl, 20 mM imidazole, adjusted to pH 8.0). The elution buffer in three-fold volume of the column volume _{(50 mM NaH 2 PO 4,} 300 mM NaCl, 250 mM imidazole, pH 8.0 adjusted) was carried out elution of MBP-His-GFP protein. Group 2 was suspended in column buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM ETDA, adjusted to pH 8.0). The supernatant was obtained and loaded onto an amylose affinity column. Washed with column buffer and eluted with column buffer containing 10 mM maltose. After purification of the protein, all fractions were confirmed by 8% SDS-PAGE analysis (Fig. 9).

<4-3> Characteristics of amylose magnetic beads

The morphology and electronic mapping of amylose magnetic beads were observed with a transmission electron microscope (TEM, JEM-2010F, JEOL, Tokyo, Japan). The chemical composition of the sample was measured with an EDX spectrometer. 10 shows TEM images, electron mapping, and EDX spectra of amylose magnetic beads. The prepared amylose beads are spherical in shape and have a diameter of about 1.5 탆. From the TEM and electron mapping it can be clearly observed that the iron oxide nanoparticles are located in the amylose beads. It has also been found that C and O are located in beads that indicate the presence of carbohydrates such as amylose molecules.

In addition, the inclusion compound type amylose beads collecting the object molecules prepared by the method of manufacturing the amylose beads according to the present invention can be utilized as a carrier for drug or functional food material. In particular, micro-magnetic amylose beads prepared using magnetic beads as object molecules can be used for the separation, purification and concentration of maltose binding protein (MBP) fusion proteins. Therefore, the amylose beads produced by the method of manufacturing amylose beads according to the present invention can be utilized in technical fields such as medicine and food field, biosensor, cell study, and biomedical materials have.

In one embodiment of the present invention, amylose magnetic beads were prepared by enzymatic synthesis of amylose using amylosecracase and iron oxide nanoparticles as object molecules. The amylose magnetic beads have a nearly perfect spherical shape with a diameter of 2.09 X 0.42 μm. The prepared amylose magnetic beads simultaneously have pure B-type amylose and Fe ₃ O ₄ crystals. The amylose magnetic beads exhibit excellent magnetic properties and redispersibility due to simple shaking, making it possible to be used for magnetic separation applications. Amylose magnetic beads have a high degree of specificity for MBP-fusion proteins and safety for several reuse circulations including regeneration and isolation. To demonstrate practical application, MBP-fused GFP proteins were successfully isolated and purified from E. coli cell lysates using amylose magnetic beads with high purification capacity (72 [mu] g / mg). Therefore, the present invention provides a simple method for manufacturing amylose magnetic beads that can be applied to various fields such as protein separation and purification, biosensors, and actuators.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, This is possible.

Claims

A step of reacting the amylose synthesizing enzyme with at least one substrate selected from glucose, oligosaccharide and sucrose and a magnetic nanoparticle as a guest molecule to prepare an amylose magnetic bead complex Wherein the magnetic beads are made of amylose.

The method according to claim 1, wherein the amylose synthase comprises at least one selected from the group consisting of amylosucrase, phosphorylase, starch synthase, amylase, and D-enzyme Wherein the magnetic beads are used as the magnetic beads.

3. The method of claim 2, wherein the amylose synthase is amylosucrase.

The method of claim 1, wherein the magnetic nanoparticles are iron oxide nanoparticles.

(a) preparing an amylose magnetic bead complex by reacting amylose synthase with at least one substrate selected from glucose, oligosaccharide, and sucrose and a magnetic nanoparticle as a guest molecule; step;
(b) binding the target protein to the amylose magnetic bead complex; And
(c) separating and purifying the amylose magnetic bead conjugate to which the target protein is bound, using the amylose magnetic beads.

6. The method according to claim 5, wherein the amylose synthase is at least one selected from the group consisting of amylosucrase, phosphorylase, starch synthase, amylase, and D-enzyme Wherein the target protein is purified by using an amylose magnetic bead.

[Claim 7] The method according to claim 6, wherein the amylose synthase is amylosucrase.

The method of claim 5, wherein the magnetic nanoparticles are iron oxide nanoparticles.

[6] The method of claim 5, wherein the target protein is MBP (maltose binding protein) -binding protein (MBP-tagged protein).

[6] The method of claim 5, wherein the step (c) comprises: separating the target protein from the amylose magnetic beads by adding a competitive binder to the target protein on the surface of the amylose. &Lt; / RTI >