KR101809929B1

KR101809929B1 - Calcium binding proteins and coacervate formed from calcium binding proteins

Info

Publication number: KR101809929B1
Application number: KR1020150064348A
Authority: KR
Inventors: 최유성; 손채연; 차형준; 반소영
Original assignee: 충남대학교 산학협력단
Priority date: 2015-05-08
Filing date: 2015-05-08
Publication date: 2017-12-18
Also published as: KR20160131586A

Abstract

The present invention relates to a calcium binding protein and a coacervate formed thereby. The novel recombinant calcium binding protein of the present invention can bind to calcium ions and is excellent in the ability to form a coacervate, so that calcium binding protein and coacervate using the same can be industrially usefully used.

Description

Calcium binding proteins and coacervate formed from calcium binding proteins < RTI ID = 0.0 >

The present invention relates to a calcium binding protein and a coacervate formed thereby.

The coacervate is a kind of colloidal material which is formed by the ionic polymer electrolyte itself or the cationic polymer electrolyte and the anionic polymer electrolyte under a specific condition. When the coacervate is formed, the absorbance of the solution increases, Which is separated from the external solution. The coacervate may be formed by the interaction of the dissolved protein with an electrolyte salt ion or alcohol, or by mixing with a polymer electrolyte having an opposite property to a specific protein. Coacervate is also used to formulate foods and cosmetics, and it is also used to immobilize functional substances such as drugs, enzymes, cells, and food additives in microcapsules due to low surface tension.

Calcium-binding protein is one of the components of various forms of biomineral such as bones, teeth, and skin, which are living organisms in nature. It is used to adjust the physical, mechanical and biological properties of biomineral which can not be expressed by inorganic materials alone, to meet the purpose of life. Calcium-binding proteins are known to bind to calcium ions and form complexes. In general, negative amino acids are the main constituents of proteins. Therefore, the calcium binding protein is likely to have the characteristics of an anionic polyelectrolyte. In addition, it can be used with biomaterials for bone regeneration such as bones and teeth composed of calcium phosphate and calcium carbonate to efficiently implant and fill calcium phosphate and calcium carbonate-based biomaterials in damaged or missing bone regions You can help.

However, until now, calcium binding protein has been obtained mainly by extraction from living organisms in a small amount, and biochemical studies have been conducted to a certain extent, but the mass production is relatively small in order to be utilized industrially. In addition, the results of formation of coacervate using calcium binding protein have hardly been reported. Therefore, there is a need for a new method for mass production of calcium binding protein and coacervate using the same.

The present inventors studied a calcium binding protein and a method for mass production of a coacervate using the same, and confirmed a novel recombinant calcium binding protein and completed the present invention.

Accordingly, an object of the present invention is to provide a calcium binding protein comprising a polypeptide represented by SEQ ID NO: 1 and a coacervate containing the same.

In order to accomplish the above object, the present invention provides a calcium binding protein comprising the polypeptide of SEQ ID NO: 1.

The present invention also provides a composition for forming a coacervate comprising a polypeptide represented by SEQ ID NO: 1.

The present invention also provides a coacervate comprising the polypeptide of SEQ ID NO: 1.

The present invention also provides a method for producing a calcium binding protein comprising the step of preparing a transformed microorganism with a polynucleotide encoding a polypeptide represented by SEQ ID NO: 1.

The novel recombinant calcium binding protein of the present invention can bind to calcium ions and is excellent in the ability to form a coacervate, so that calcium binding protein and coacervate using the same can be industrially usefully used.

Brief Description of the Drawings Fig. 1 is a schematic diagram showing a recombinant calcium binding protein expression vector pGG.
Figure 2 shows the result of SDS-PAGE analysis of the purified recombinant calcium binding protein (M, protein marker: BL & IS, negative control of BL21 (DE3) cells; S, water soluble fraction of GG1234; FT, W, wash fraction; E, refined GG1234).
FIG. 3 is a graph showing the calcium binding ability of GG1234 by SDS-PAGE and Stains-all staining (GG: GG1234).
FIG. 4 is a graph showing the results of confirming the calcium binding ability of GG1234 according to GG1234 concentration. FIG.
FIG. 5 is a graph showing the results of confirming the coacervation-forming ability of GG1234 according to the pH change through the change in absorbance.
Fig. 6 is a diagram showing the result of visually confirming the formation of coacervate in GG1234. Fig.
FIG. 7 is a graph showing the result of the determination of the formation of complex coacervate by the mixture of GG1234 and fp-151 through the change in absorbance (GG-151 coacervate: complex coacervate of GG1234 and fp151).
FIG. 8 is a diagram showing the result of microscopic observation of the complex type of coacervate. FIG.

The present invention provides a protein for calcium binding, which is composed of the polypeptide represented by SEQ ID NO: 1.

The polypeptide of SEQ ID NO: 1 of the present invention is a recombinant protein having calcium-binding ability capable of binding calcium, and can effectively form coacervate and can be mass-produced in a water-soluble form, have.

The polypeptide represented by SEQ ID NO: 1 is a sequence registered with Genebank XP_001234449, which is encoded by a gene derived from Gallus gallus, and may also include mutants thereof to the extent that calcium binding ability is maintained. By mutant is meant addition of an additional amino acid sequence or substitution of some amino acid at the carboxyl terminus or amino terminus.

In this case, the mutant of the polypeptide represented by SEQ ID NO: 1 may include, without limitation, a polypeptide having a functional equivalent thereto, and may be a polypeptide having at least 70%, preferably at least 80%, more preferably Refers to having at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, still more preferably 99% sequence identity.

The polypeptide may be mass-produced in E. coli using a codon-optimized DNA sequence of a part of the mRNA sequence, and the mass-produced recombinant polypeptide is over-expressed in a water-soluble manner.

The term codon optimization refers to modifying a rarely existing codon in a host cell to a frequently occurring codon in a nucleic acid encoding a heterologous recombinant protein to produce a heterologous recombinant protein more efficiently in a host cell, Refers to the expression of a recombinant nucleotide after transformation with a nucleic acid. Such optimization in heterologous expression systems can improve the host's ability to produce foreign proteins.

The protein for calcium binding in the present invention is not limited thereto, but it can be inserted into a conventional vector which is designed for expressing an external gene, and can be mass-produced by a genetic engineering method. The vector may be suitably selected according to the type and characteristics of the host cell for producing the protein, or may be newly produced. A method of transforming the vector into a host cell and a method of producing a recombinant protein from the transformant can be easily carried out by a conventional method. Methods for selecting, producing, transforming and expressing recombinant proteins described above can be easily performed by those skilled in the art, and some modifications are also included in the present invention in ordinary methods.

The polypeptide of SEQ ID NO: 1 of the present invention forms a large amount of complex when bound to calcium ions and can form a precipitate. Accordingly, the polypeptide of SEQ ID NO: 1 of the present invention constitutes a protein for calcium binding for use as a calcium binding protein.

The protein for calcium binding comprising the polypeptide represented by SEQ ID NO: 1 and the mutant retaining its function may be characterized by containing a large amount of anionic amino acid.

The term coacervate is a type of colloidal material that is formed when anionic polyelectrolyte and cationic polyelectrolyte are mixed under specific conditions and refers to a substance that exists in the form of a spherical sphere in solution and exists separately from the external solution.

The coacervate of the present invention may be a calcium binding protein consisting of the polypeptide represented by SEQ ID NO: 1 or a colloid formed by binding the recombinant calcium binding protein and a cationic polymer, preferably a water-soluble solvent, Methanol, ethanol, propanol, acetone, acetic acid aqueous solution, and more preferably in an aqueous solution of sodium acetate.

In the case of producing the coacervate in the solvent, the addition amount of the calcium binding protein and the cationic polymer is not limited thereto, but may be preferably 0.001 to 100% (w / v) relative to the volume of the entire solvent, more preferably 0.01 To 30% (w / v).

The composition for forming a coacervate may further include an electrolyte or a polymer having various pHs and concentrations capable of forming a coacervate by mixing with the polypeptide represented by SEQ. ID. NO. 1, and the optimum pH is preferably pH 2.0 to pH 10.0 Preferably from pH 2.0 to pH 6.0, more preferably from pH 2.5 to pH 5.5.

That is, the calcium binding protein represented by SEQ ID NO: 1 of the present invention can form a coacervate by binding to an electrolyte salt or a cationic polymer in an acidic pH range. A coacervate formed by binding calcium binding protein to an electrolyte salt is called simple coacervate and a complex coacervate formed by binding with a cationic polymer is defined as a coacervate.

The composition for forming a coacervate according to the present invention may further comprise at least one cationic polymer in addition to the polypeptide represented by SEQ ID NO: 1, which may form a complex coracervate.

The cationic polymer may include, but is not limited to, a polymeric substance capable of forming a coacervate by binding with an anionic protein, a calcium binding protein composed of a polypeptide represented by SEQ ID NO: 1, More preferably a polymer having a pI value of 6 to 12, more preferably a polymer having a pI of 8 to 11, which is higher than the pI (Isoelectric point) of a calcium binding protein composed of a polypeptide or a variant thereof. When the pI value is more than or less than the above-mentioned pI value, it is difficult to form a coacervate, so that it is preferable to use the cationic polymer within the above-mentioned pI range.

According to the above production method, calcium binding protein can be mass-produced in a water-soluble form.

More specifically,

A step of codon-optimizing the mRNA sequence of the polypeptide represented by SEQ ID NO: 1 and a restriction enzyme site at both ends of the DNA of the optimized calcium binding protein and cloning into an expression vector;

Preparing a transformant into which the expression vector is introduced;

And purifying the recombinant calcium binding protein prepared by culturing the transformant.

The term vector is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. The vector can replicate the DNA and be independently reprogrammed in the host cell. The term expression vector refers to a recombinant DNA molecule comprising the desired coding sequence and a suitable nucleic acid sequence necessary for expressing the coding sequence operably linked to the particular host organism. The expression vector may preferably comprise one or more selectable markers, and such markers are nucleic acid sequences that are capable of being selected generally by a chemical method, which can distinguish the transformed cells from non- This is all the genes that are present. Examples include, but are not limited to, antibiotic genes such as kanamycin, bleomycin, chloroampinicol, and ampicillin.

The present invention also provides a transformant transformed with said vector.

The term transformant refers to the introduction of a new target polynucleotide into a host cell, and the host cell may include, but is not limited to, E. coli, yeast, animal, plant cell or insect cell.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Preparation of Recombinant Calcium Binding Protein

Recombinant calcium binding protein was prepared using the sequence of SEQ ID NO: 1 registered with Genebank XP_001234449. More specifically, in order to express the sequence of SEQ ID NO: 1 in a large amount in E. coli, a codon-optimized DNA sequence of a part of the mRNA sequence was chemically synthesized. NdeI and XhoI restriction sites were added to both ends of the optimized DNA and cloned into an expression vector pET23b (+) containing the expression promoter T7 to prepare a recombinant protein expression vector. For the purification of the recombinant protein, a histidine tag (HHHHHH) consisting of six histidine sequences at the C-terminus was included in the expression vector, and an ampicillin resistance gene was included so as to confirm gene introduction. FIG. 1 shows a schematic diagram of the prepared protein expression vector pGG, and the recombinant protein thus produced was named GG1234.

For mass production of GG1234, recombinant protein expression vector pGG was introduced into Escherichia coli BL21 (DE3) at 42 DEG C for 1 minute and 30 seconds with thermal shock. Since pGG is a resistance-resistant vector in ampicillin antibiotics, it was cultured in LB-agar medium supplemented with ampicillin, and the transformed E. coli into which the vector was introduced was selected. The selected transformant 50㎍ / mL Amphitheater is added a conventional 37 ℃, the absorbance of the culture solution by shaking culture in LB medium cylinder of an inducer when the (OD ₆₀₀₎ reaches approximately 0.8 ~ 0.9 IPTG (isopropyl-β -D-thiogalactopyranoside, 1 mM) was added to induce the expression of GG1234. After addition of IPTG, the cells were cultured at 20 ° C for 20 hours. The cultured cells were centrifuged at 4000 rpm for 10 minutes, and the supernatant was removed and the cells were recovered. The recovered cells were suspended in a solution for cell disruption (50 mM sodium phosphate buffer, pH 7, 10 mM imidazole) and then disrupted using an ultrasonic disintegrator. The disrupted cells were divided into a water-soluble fraction and an insoluble fraction, and each was analyzed using conventional SDS-PAGE. Further, a water-soluble fraction of the protein was applied to a column packed with nickel resin to allow binding of the protein to the column, and a protein not bound to the column with a washing buffer (50 mM sodium phosphate buffer, 20 mM imidazole, pH 7.0) I washed it. Elution of the protein from the column was carried out using elution buffer (50 mM sodium phosphate buffer, 300 mM imidazole, pH 7.0), and water and protein components remaining in the protein aqueous solution were removed by dialysis using water Finally, the purified protein was produced. The soluble fraction of E. coli expressing the recombinant calcium binding protein and the recombinant calcium binding protein purified by the nickel column chromatography were analyzed by SDS-PAGE and the results are shown in FIG.

As shown in FIG. 2, the recombinant protein was overexpressed and detected as water-soluble, and it was confirmed that the recombinant protein GG1234 could be mass-produced in a water-soluble form and easily separated and purified.

Example 2. Confirmation of Calcium Binding Capacity of Recombinant Calcium Binding Protein GG1234

It was confirmed whether the protein isolated and purified in Example 1 exhibited the calcium binding protein-binding calcium binding property.

2.1 Analysis by stains-all staining

Since the calcium binding protein contains many anionic amino acids, it is known that calcium binding ability can be indirectly confirmed by Stains-all staining. This is because a cationic carbocyanine dye binds to the calcium-binding site of the protein to form a dye-protein complex. This complex has a specific absorbance in the wavelength range of 600 to 615 nm, and a dyeing reagent was applied , While most proteins that do not have calcium binding ability do not form complexes, so they use the principle of staining proteins with red color. FIG. 3 shows the results of SDS-PAGE and Stains-all staining for the calcium binding of GG1234 protein of Example 1. As a negative control, BSA protein was used.

As shown in FIG. 3, it was confirmed that GG1234 protein was stained with blue color unlike BSA which is a negative control group. As a result, it was confirmed that GG1234 protein is a protein likely to bind calcium.

2.2 Confirmation of formation of precipitate of GG1234 and calcium ions

Since the GG1234 protein was expected to have the ability to bind calcium, a direct mixing of the GG1234 protein with calcium ions was performed and the absorbance was measured at a wavelength of 600 nm to confirm this directly. The calcium ion was fixed at 10 mM, the concentration of GG1234 protein was varied, and the absorbance was measured. The result is shown in FIG.

As shown in FIG. 4, the GG1234 protein reacted with calcium ions to form a large number of complexes, thereby forming many precipitates. As a result, it was confirmed that GG1234 was a calcium binding protein having calcium binding ability.

Example 3. Coacervate formation of GG1234 protein

Since it was confirmed that the recombinant protein GG1234 of Example 1 was a calcium-binding protein, it was confirmed by observation of absorbance and microscopic observation whether coacervate was formed when sodium acetate buffer and pH were added at various concentrations. The sodium acetate buffer solution was added at a concentration of 200 mM, and the absorbance was measured at a wavelength of 600 nm while changing the pH to about 3 to 5. [ The results are shown in Fig. 5 and Fig.

As shown in FIG. 5, the highest absorbance was observed at pH 3.75, and it was confirmed through microscopy that a prototype having a size of about 2 to 3 μm was formed. As shown in FIG. 6, addition of 1 to 200 mM sodium acetate buffer at pH 3.75, which exhibits the highest absorbance, was visually confirmed that spherical coacervate was effectively formed.

Example 4. Complex Coacervate Formation Using Recombinant Calcium Binding Protein GG1234 and the Cationic Polymer Protein fp-151

The recombinant calcium binding protein GG1234 and the representative cationic protein polymer fp-151 (SEQ ID NO: 2) were mixed at a ratio of 1: 1 (w / w) and coacervate was formed Was confirmed by turbidity change. The turbidity change was measured by measuring the absorbance at 600 nm, and the results are shown in FIG.

As shown in FIG. 7, turbidity changes were observed by mixing GG1234 and fp-151, which is due to the formation of complex coacervate. The complexed coacervate formed was named GG-151. The type of coacervate at pH 4 showing the highest absorbance and pH 5 and pH 6 showing low absorbance was observed under a microscope, which is shown in FIG. As shown in FIG. 8, it was confirmed that the highest concentration of complex coacervate GG-151 was formed at pH 4 showing the highest absorbance.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Calcium binding proteins and coacervate formed from calcium binding proteins <130> 1-26p <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 284 <212> PRT <213> Gallus gallus <400> 1 Met Gln Glu Leu Val Ala Thr Glu Arg Arg Glu Val Ala Thr Met Gln 1 5 10 15 Glu Leu Val Ala Thr Trp Lys Asp Glu Val Ala Met Met Arg Gly Glu 20 25 30 Val Ala Thr Trp Lys Asp Glu Val Ala Met Met Arg Gly Glu Val Ala 35 40 45 Thr Trp Lys Asp Glu Val Ala Thr Val Gly Glu Glu Val Ala Thr Trp 50 55 60 Lys Glu Leu Gly Ala Gln Lys Ala Ala Val Gln Leu Glu Lys Met Glu 65 70 75 80 Asp Asn Gly Asp Asn Gly Asp Ser Gly Asp Asn Gly Asp Leu Gly Asp 85 90 95 Asn Gly Asp Leu Gly Asp Asn Gly Asp Ser Glu Asp Ser Gly Asp Cys 100 105 110 Gly Asp Val Gly Asp Asn Gly Asp Ser Gly Asp Cys Gly Gly Ser Gly 115 120 125 Asp Asn Gly Ala Asn Gly Asp Asn Gly Asp Arg Gly Asp Asn Gly Asp 130 135 140 Ser Gly Asp Ser Gly Asp Cys Gly Asn Gly Arg Asp Ser Gly Ala Asn 145 150 155 160 Gly Asp Leu Gly Asp Tyr Gly Asp Ser Gly Gly Ser Gly Asp Asn Gly 165 170 175 Asp Arg Asp Ser Gly Asp Ser Gly Asp Asn Gly Ala Asn Gly Asp Cys 180 185 190 Glu Asp Asn Gly Asp Ile Arg Asp Ile Gly Asp Ile Gly Asp Ser Gly 195 200 205 Asp Thr Gly Asp Asn Arg Asp Ser Gly Val Asn Gly Asp Ser Gly Asp 210 215 220 Ser Gly Ala Asn Gly Asp Leu Gly Asp Asn Gly Asp Ile Gly Asp Ile 225 230 235 240 Gly Asp Ser Gly Asp Thr Gly Asp Asn Arg Asp Ser Gly Val Asn Gly 245 250 255 Asp Ser Gly Asp Asn Gly Ala Asn Gly Asp Ser Gly Asp Asn Gly Asp 260 265 270 Ile Gly Asp Ser Glu Asp Asn Gly Asp Val Gly Thr 275 280 <210> 2 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> fp-151 <400> 2 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala 20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro 35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu 50 55 60 Gly Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser 65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys 85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly 100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr 115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr 130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 180 185 190 Pro Thr Tyr Lys 195

Claims

1. A composition for calcium binding comprising a polypeptide represented by SEQ ID NO: 1.

1. A composition for forming a coacervate comprising a polypeptide represented by SEQ ID NO: 1.

The composition for forming a coreacebate according to claim 2, further comprising a cationic polymer.

4. The composition of claim 3, wherein the coacervate is a complex coacervate.

1. A coacervate comprising a polypeptide represented by SEQ ID NO: 1.

Adding the polypeptide of SEQ ID NO: 1 to sodium acetate buffer pH 3 to 5.

Preparing a mixture by mixing the polypeptide of SEQ ID NO: 1 with a cationic polymer; And
And adding the mixture to a sodium acetate buffer solution at pH 3 to 6. The method of claim 1, wherein the complex coacervate is sodium chloride.