KR100388935B1 - Recombinant βig-h3 protein and producing method thereof - Google Patents

Abstract

The present invention relates to a method for producing the recombinant protein and full-length βig-h3 protein using a recombinant protein and E. coli as a host, including all or part of a native βig-h3 protein to support cell adhesion and proliferation. It is water-soluble, so that it is easy to separate and purify, and can obtain a large amount of protein for a part of βig-h3 which is excellent in cell adhesion and diffusion effect, and supports cell adhesion and diffusion in the same way as the original form made in mammalian cells. Can produce a large amount of recombinant βig-h3 protein.

Description

Recombinant βig-h3 protein and producing method

The present invention relates to a recombinant protein comprising all and part of the domain of the βig-h3 protein and a method for producing the same, and more specifically, to a cell adhesion and diffusion prepared by using E. coli as a host. The present invention relates to a recombinant protein comprising all and part of a domain thereof and a method for producing the same.

βig-h3 was first identified by Skonier et al. by constructing a cDNA library from adenocarcinoma cell line (A549) of human lungs treated with TGF-β and screening the library.

βig-h3 is composed of 683 amino acids and encodes a protein having an Arg-Gly-Asp (RGD) sequence that acts as a ligand recognition site for various integrins at the carboxy terminus and a secretory sequence at the amino terminus.

There are four repeat domains called fasciclin-I in this protein, and some secreted proteins or membrane proteins from several species, including mammals, insects, sea urchins, yeast and bacteria, are highly homologous. It turns out to have a motif.

In many cell lines, the expression of βig-h3 mRNA and protein is increased by TGF-β. This fact is believed to be involved in mediating some of the signals of TGF-β because βig-h3 genes are induced in several cell systems whose proliferation is affected by TGF-β.

By contrast, βig- It has been reported that h3 expression is reduced and regulated.

As described above, βig-h3 has been reported to act as an important factor in the interaction and morphogenesis between the extracellular matrix and the cells in various tissues.

Βig-h3 is a cell adhesion molecule and is known to mediate cell adhesion and proliferation. For example, purified βig-h3 protein promoted the adhesion and proliferation of dermal fibroblasts, while inhibiting the adhesion of A549, HeLa and WI-38 cells in serum-free medium.

In particular, it is known to inhibit the growth, colony formation and emergence of tumor cells. Transfection of βig-h3 expression plasmids into Chinese Hamster Ovary (CHO ") cells forms tumors in nude mice. It is disclosed in US Pat. Nos. 5,599,788 and 5,714,588 that the ability of these cells to significantly decrease.

Also disclosed in these patents are methods for promoting the spread and adhesion of cells, especially fibroblasts, to wounds by contacting the wound with an effective amount of βig-h3 to promote healing to promote rapid wound healing. .

As described above, βig-h3 is a substance that is expected to be useful in various ways, but only a very small amount can be collected in vivo.

In order to solve this problem, a method of producing by expressing in a eukaryotic cell system by genetic recombination has been disclosed in the above patents. However, when expressed in the eukaryotic cell system, the cells producing βig-h3 are much slower than the cells which do not produce, so it is difficult to secure a sufficient number of cells, resulting in the production of βig-h3 obtained in the eukaryotic cell system. There is a problem that the mass production is difficult because of the small number.

In addition, the above patents use ammonium sulfate precipitation and gel filtration column chromatography to purify the protein, but how pure protein can be obtained by other kinds of chromatography alone. It is not mentioned.

Accordingly, the present inventors have succeeded in mass-expressing and purifying recombinant proteins including all and part of the domain of βig-h3 protein using E. coli as a host, and the present invention was confirmed by confirming that the recombinant proteins support cell adhesion and proliferation. Was completed.

An object of the present invention is to provide a method capable of easily producing a purified large amount of recombinant βig-h3 by producing a recombinant protein comprising all and part of the domain of βig-h3 protein using E. coli as a host.

In addition, an object of the present invention is to provide a recombinant protein that can be produced in large quantities in E. coli and has a water solubility and is easy to be separated, while exhibiting the same cell adhesion and diffusion effects as recombinant βig-h3.

1 is a diagram showing the structure of the recombinant βig-h3 protein,

2 is a diagram of recombinant proteins corresponding to the fourth repeat domain of βig-h3 protein,

3aIs Recombination The photo shows the results of polyacrylamide electrophoresis of βig-h3 proteins,

1: Hisβ-a

2: Hisβ-b

3: Hisβ-c

4: Hisβ-d

Figure 3b is a photograph showing the results of polyacrylamide electrophoresis of Hisβ-a and Hisβ-b proteins,

Figure 3c is a result of analysis by SDS-PAGE after crosslinking Hisβ-a and Hisβ-b with glutaraldehyde,

◀: Multiple conjugate

Figure 3d is an electron micrograph showing the fiber structure of the recombinant βig-h3 shown in the electron microscope after negative staining,

4A to 4C are micrographs and graphs showing the results of experiments on adhesion and diffusion of CHO cells to a surface coated with BSA, Hisβ-b or FN,

5Is Recombination Bar graph showing the results of confirming the specificity of the cell adhesion activity mediated by βig-h3,

6aIs Recombination It is a bar graph showing the result of confirming the receptor of the cell adhesion mediated by βig-h3,

Figure 6b is a bar graph showing the results of experiments in the reactivity of the recombinant proteins Hisβ-g and Hisβ-e with integrin,

7Silver recombination It is a bar graph showing the result of analyzing the divalent cation sensitivity to the cell adhesion mediated by βig-h3.

8aWow8bN terminal is deleted Bar graph showing the results of experiments on adhesion and diffusion of CHO cells to substrates coated with βig-h3 protein,

9a is a photograph showing the result of ND-PAGE analysis after crosslinking Hisβ-e with glutaraldehyde,

1: Hisβ-b

2: Hisβ-e

3: BSA

9b is a photograph showing the result of SDS-PAGE analysis after crosslinking Hisβ-e with glutaraldehyde,

◀: Dimer

10A to 10C are graphs showing the results of comparing the adhesion of CHO cells to surfaces coated with Hisβ-b and Hisβ-e,

10B is a graph showing the diffusion area of CHO cells on the surface coated with Hisβ-b and Hisβ-e,

11A and 11B are graphs showing the binding of Hisβ-b protein and Hisβ-e protein to immobilized extracellular matrix protein,

12 is a diagram of a mammalian expression vector for βig-h3,

13A and 13B are bar graphs showing the results of experiments on cell adhesion and proliferation of βig-h3 expressed by the expression vector of FIG. 12.

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, in the present invention, the recombinant protein comprising a part of the native βig-h3, the recombinant consisting of the amino acid sequence and the selection marker of the deletion of all or a terminal portion of amino acids 1-653 at the position of SEQ ID NO: 2 Provided are a recombinant protein comprising an amino acid sequence and a selection label identical or substantially similar to positions 237-377 or 498-637 of protein and SEQ ID NO: 2.

In particular, the recombinant protein comprising the amino acid sequence of 237-377 of βig-h3 protein comprises the second repeat domain of βig-h3 protein, and the recombinant protein comprising the amino acid sequence of 498-637 represents the fourth of βig-h3 protein. Recombinant protein comprising a repeating domain is the same or better than the full-length βig-h3 protein cell adhesion and proliferation ability, water soluble and easy to isolate.

In addition, the present invention provides a step of preparing a cDNA fragment comprising a nucleotide sequence encoding a selectable sequence and the base sequence encoding all or part of the terminal of all the amino acids of positions 1-653 in SEQ ID NO: 2, forming an expression vector into which the cDNA fragment is inserted, transfecting the expression vector to transform E. coli, expressing the protein in the transformed E. coli, and expressing the expressed protein using a selection label. It provides a method for producing a recombinant protein, characterized in that for producing a large amount of protein supporting the cell adhesion and diffusion, including the step of purifying. Escherichia coli transformant E. coli BL21 / Hisβb used in the above production method was deposited on April 25, 2000 to the Biotechnology Research Institute Gene Bank (Accession Number: KCTC 18008P).

In addition, the present invention is to prepare a cDNA fragment comprising a nucleotide sequence encoding the amino acid of position 237-377 or 498-637 and SEQ ID NO: 237-377 or SEQ ID NO: 2, the expression vector into which the cDNA fragment is inserted Forming a cell, transfecting the expression vector to transform E. coli, expressing the protein in the transformed Escherichia coli, and purifying the expressed protein using a selection label. It provides a method for producing a recombinant protein, characterized in that for producing a large amount of protein supporting the adhesion and diffusion. The E. coli BL21 / Hisβ-g and E. coli BL21 / Hisβ-e were transformed into the Genetic Bank of Biotechnology Research Institute on April 25, 2000 (accession number: KCTC 18010P and accession number: KCTC). 18009P).

By producing a recombinant protein using E. coli as a host as described above it is possible to produce a large amount of useful substances having the same or similar functions as the original βig-h3 protein.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples.

Example 1 Production of Recombinant βig-h3 Protein in Escherichia Coli

In order to produce recombinant βig-h3 protein, four expression vectors, pHisβ-a, pHisβ-b, pHisβ-c and pHisβ-d, were prepared as follows.

As a vector for expression of recombinant βig-h3 protein, pET-29 β (+) vector (Novagen) was used.

βig-h3 cDNA cloned into pBluescript (pBS βig-h3) was cleaved into Nde I and Bgl II and converted into blunt ends, and the fragment was subcloned into EcoR V and EcoR I sites of pET-29 β. PHisβ-a was prepared.

pHisβ-b is restrained Asp 718 from the βig-h3 cDNA - was prepared by inserting a EcoR Ⅴ of the Bgl Ⅱ fragments pET-29β and EcoR Ⅰ site.

pHisβ-c was prepared by introducing an Nco I fragment of 1351 nucleoids excised from βig-h3 cDNA into the Nco I site of pHisβ-b.

Hisβ-d was prepared by cutting 3′-fragments of Hisβ-c vector into Aoc I and Not I to make blunt ends and then self ligation.

In order to purify the recombinant proteins using Ni-NTA resin, six His-tags were linked to the ends of the recombinant protein. In E. coli, full-length recombinant proteins can be derived but cannot be purified using Ni-NTA resin because the C terminus is cleaved and His-label attached to the C terminus is lost. Thus, in the present invention, 30 amino acids were cut from the C terminus of the recombinant protein and linked to 6 His-labels.

Clones in which DNA fragments were inserted in the right direction were selected, and nucleotide sequences were checked to confirm whether mutations or deletions occurred during the cloning process.

E. coli BL21 (DE3) was used as a host for expressing the vectors and transformed strains were kanamycin (37 ° C., 50 μg / ml) until the optical concentration (OD) at 595 nm was 0.5-0.6. cultured in LB medium containing kanamycin).

Among the transformed strains, E. coli BL21 (DE3) transformed with pHisβ-b was named E.coli BL21 / Hisβ-b and was deposited on April 25, 2000 at the Biotechnology Research Institute Gene Bank. Number: KCTC 18008P).

Βig incubated at 37 ° C. for 3 hours with 1 mM isoproyl-β-D-(-)-thiogalactopyranoside (hereinafter referred to as "IPTG"). -h3 protein was induced and expressed.

After expressing βig-h3, the E. coli pellets were purified by 50 mM Tris hydrochloric acid (Tris-HCl, pH 8.00), 100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethane-sulphate. It was resuspended in cell lysis buffer consisting of phenylmethane sulfonyl fluoride (hereinafter referred to as "PMSF") and 0.5 mM DTT and sonicated. This procedure was repeated five times.

After sonication, centrifugation was carried out, and after centrifugation, insoluble bodies containing βig-h3 were separated by 8 M containing 20 mM tris hydrochloric acid (pH 7.8), 0.5 M sodium chloride, and 5 mM imidazole. It was dissolved in denaturing buffer of urea. After dissolution, the denatured protein was purified using Ni-NTA resin (Qiagen) according to the resin manufacturer's instructions.

The recombinant protein was eluted with a 200 mM imidazole solution, and the eluted recombinant protein was renaturated by dialysis in a high concentration to low concentration urea in 20 mM Tris hydrochloric acid buffer solution containing 50 mM sodium chloride.

1 shows a diagram of the βig-h3 recombinant proteins as described above. The hatched and cross hatched cells in FIG. 1 represent highly conserved sequences of each of the repeating domains I, II, III and IV, and the RGD motif is shown as blank.

As can be seen in FIG . 1 , the recombinant protein of the present invention does not have 30 amino acids from the C terminus. In addition, at pHisβ-a and pHisβ-b, N-terminal 174 amino acids and 68 amino acids were deleted, respectively, and pHisβ-d lacked the RGD sequence.

As a result of quantifying the recombinant protein prepared by the method as described above, in the case of pHisβ-b, 9 mg per liter of bacterial culture solution could be separated and purified.

Example 2 Production of Recombinant Proteins for Second and Fourth Repeat Domains

A polymerase chain reaction (PCR) was used to make fragments of βig-h3 cDNA encoding amino acids 237-377 and 498-637 in SEQ ID NO: 2, respectively, and the fragments were cloned into EcoR V and pET-29β. Cloning to Xho I site, pHisβ-g and pHisβ-e were prepared. At this time, in order to purify the synthesized protein using Ni-NTA resin, nucleotides encoding six His-labels at the C terminus were linked as in Example 1.

As a host for expression of the vector was used to E.coli BL21 (DE3), E.coli BL21 transformed with a pHisβ-e (DE3) was named as E.coli BL21 / Hisβ-e-g to pHisβ Transformed E. coli BL21 (DE3) was named E. coli BL21 / Hisβ-g and deposited with the Biotechnology Research Institute Gene Bank on April 25, 2000 (Accession Number: KCTC 18009P and Accession Number: KCTC 18010P). . The transformants were incubated in LB medium containing kanamycin at 37 ° C. and 50 μg / ml until the optical concentration (OD) at 595 nm was 0.5-0.6. Hisβ-e and Hisβ-g proteins were induced by expression of 1 mM IPTG at 37 ° C. for 3 hours, and then the E. coli precipitate was purified by 50 mM Tris hydrochloric acid (pH 8.0), 100 mM sodium chloride, 1 mM EDTA, 1% Triton. It was resuspended in cell lysis buffer consisting of X-100, 1 mM PMSF and 0.5 mM DTT and sonicated. This procedure was repeated five times.

Supernatants obtained after sonication and centrifugation were purified using Ni-NTA resin (Qiagen) according to the resin manufacturer's instructions. Unlike other Hisβ constructs expressed in the insoluble fraction, Hisβ-e and Hisβ-g were purified without urea because they were expressed in the soluble fraction.

A diagram of the recombinant Hisβ-e and Hisβ-g proteins prepared as above is shown in FIG. 2 . As a result of quantification of the purified synthetic protein, 35 mg of protein per liter of the bacterial culture solution was obtained.

Experimental Example 1 Analysis of Recombinant βig-h3

The recombinant proteins prepared in Example 1 were sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE) and non-denaturing polyacrylamide gel electrophoresis. , ND-PAGE "below).

The results of electrophoresis of four purified recombinant βig-h3 proteins on a 10% polyacrylamide gel are shown in FIG. 3A . In FIG. 3a , lane 1 is Hisβ-a, lane 2 is Hisβ-b, lane 3 is Hisβ-c, lane 4 is Hisβ-d, and the molecular weight is expressed in kilodaltons (kDa) on the left side of the gel. As can be seen in FIG . 3A , all four recombinant βig-h3 proteins appeared as single bands in SDS-PAGE.

The results of electrophoresis of Hisβ-a and Hisβ-b are shown in FIG. 3B . Urease, the molecular weight basis, shows 272 kDa and 545 kDa. As can be seen in Figure 3b , Hisβ-a and Hisβ-b formed a ladder shape consisting of a plurality of bands. The results were not shown, but similar results were observed in Hisβ-c and Hisβ-d.

Experimental Example 2 Crosslinking

Glutaraldehyde to βig-h3 proteins prepared in Example 1

(glutaraldehyde, Sigma Chemical Co. St. Louis. MO.) and crosslinked. Glutaraldehyde may interact strongly or weakly with proteins in aqueous solution to form oligomo or polymers and may crosslink multiple proteins in higher order complexes.

Recombinant βig-h3 protein was incubated for 5 minutes with glutaraldehyde at a concentration of 0.01-1% at 20 ° C, and the reaction was stopped by adding a staining solution for SDS-PAGE. Crosslinked βig-h3 protein was analyzed by electrophoresis on 10% SDS-PAGE gel and the results are shown in FIG. 3C . After 0.01% glutaraldehyde treatment, distinct crosslinked forms of dimer, trimer and tetramer size were observed, and after high concentration glutaraldehyde treatment, βig-h3 protein was present in high molecular weight form. Crosslinked quantitatively. The results for Hisβ-a and Hisβ-b are representatively shown in FIG. 3C , and cross-linked forms corresponding to dimers, trimers, tetramers, and multiplexers are indicated by arrows.

Hisβ-b electrophoresed with cross-linking as described above was stained with Coomassie blue, and the oligomer structure of the recombinant proteins was observed by electron microscope in FIG. 3D . All four recombinant βig-h3 formed a fibrous structure, but typically showed only results for Hisβ-b. As shown in FIG. 3D , Hisβ-b formed a thick fibrous structure in which many thin fibrils were collected.

Experimental Example 3 Cell Attachment and Diffusion Analysis

The adhesion and proliferation of CHO cells to the surface coated with Hisβ-b were investigated. The adhesion and proliferation of CHO cells to the surface coated with FN as a control and BSA as a control were investigated. Recombinant βig-h3 protein, fibronectin (FN) and BSA at concentrations of 0 μg / ml to 100 μg / ml in 96 trillion microchromator plates (Falcon, Becton-Dickinson, Mountain View. After incubation, the cells were blocked with phosphate-buffered saline containing 0.2% BSA for 1 hour at the same temperature. CHO cells were treated with trypsin and suspended in α-MEM at a concentration of 3 × 10 ⁵ cells / ml, then 0.1 ml of cell suspension was added to each bath of the plate. After incubation at 37 ° C. for 90 min, unattached cells were washed away with phosphate buffered saline. The attached cells are 3.75 mM p - acetyl-β-D- glycoside Sami Need -1 (p -nitro-phenol- N -acetyl- 1-D-glycosaminide) { hexyl sosami The first (hexosaminidase) - nitro phenol - N Substrate} and 50 mM citric acid buffer at pH 5.0 containing 0.25% Triton X-100, and incubated at 37 ° C for 1 hour. After incubation, 4 × 10 ⁴ cells were placed in 48-thick plates and attached to the substrate. Attached cells were fixed with 8% glutaraldehyde (Sigma Chemical Co., St. Louis. MO) and stained with 0.25% (w / v) crystal violet methanol solution (Sigma). Stained cell regions were measured using Image-Pro plus software (Media Cybernetics, Silver Spring, MD.) And the results are shown in FIG. 4B .

In addition, the cells attached to the surface after the culture was blocked by adding 50 mM glycine buffer solution of pH 10.4 including 5 mM EDTA. After blocking the enzyme activity, the analysis result for hexosaminidase was confirmed by measuring the absorbance at 405 nm with a Multiscan MCC / 340 microplate reader, and the result is shown in FIG. 4C . It was. The experiment was carried out 200 to 300 times for each experimental site, repeated three times and the results are expressed as the mean ± SD (standard error) of the three measurements.

4b and 4c showed that the cell adhesion and proliferation activity of βig-h3 was concentration-dependent. The results were not shown, but the results were similar to those of the human corneal epithelial cell (hereinafter referred to as HCE "). Obtained also in cells.

In addition, after the culture by coating the respective protein in 10 ㎍ / ㎖ concentration, staining is shown in Figure 4a to the result of observation under a microscope. As can be seen in Figure 4a the number of CHO cells attached to Hisβ-b protein is greater than the number of cells attached to albumin, the surface area of CHO cells attached to Hisβ-b protein is the surface of the cells attached to albumin It was wider than the area. In addition, the number and surface area of CHO cells attached to Hisβ-b protein were similar to the number and surface area of cells attached to fibronectin.

Experimental Example 4 Inhibition Analysis

In order to confirm the specificity of the cell adhesion activity of recombinant βig-h3, various chemical probes were used.

The plastic dish was coated with 10 μg / ml of each protein of Hisβ-c, Hisβ-d or FN. CHO cells were preincubated for 30 minutes in medium containing 5 mM EGTA, 100 μg / ml βig-h3, 1 mM GRGDSP, 1 mM GRGESP, 100 μg / ml pFN or 5 μl / ml rabbit anti-βig-h3 antiserum. It was. GRGDSP or GRGESP (Gibco BRL, Gaithersburg, MD, USA) was also tested to evaluate the contribution of RDGS and related sequences to cell adhesion activity. The cell suspension was transferred to a protein coated dish and incubated for 1 hour. Cells loosely bound to cells that were not bound in the experimental bath were removed by shaking, and the remaining cells were fixed with glutaraldehyde as in Experiment 3, and the attached cells were quantified and the results are shown in FIG. 5 .

As can be seen in FIG . 5 , cell adhesion to βig-h3 was almost completely inhibited by βig-h3 itself, anti βig-h3 antiserum, RGD peptide and EDTA, and partially inhibited by fibronectin and EGTA. Not inhibited by RGE peptide. Cell adhesion to fibronectin was significantly inhibited by fibronectin itself, RGD peptide and EDTA and partially inhibited by βig-h3 and EGTA, but not by RGD peptide.

These results indicate that cell adhesion to βig-h3 is specific, and it can be seen that βig-h3 interacts with RGD-dependent integrin at least in CHO cells and HCE cells.

Experimental Example 5 Receptor Identification of Cell Attachment of βig-h3

To identify receptors for βig-h3, monoclonal antibodies against various integrin subunits were investigated for their effect on the adhesion of HCE cells to surfaces coated with βig-h3.

The HCE cells were individually isolated in the presence of monoclonal antibodies against various types of integrins (Chemigon International Inc. Temecula, CA. USA) at 0.05 ml of culture solution (3 × 10 ⁵ cells / ml) at 37 ° C. Preculture for 30 minutes.

The antibodies used were as follows: α1, antiintegrin α1 subunit antibody (FB12); α2, antiintegrin α2 subunit antibody (P1E6); α3, antiintegrin α3 subunit antibody (P1B5); α4, antiintegrin α4 subunit antibody (P1H4); α5, antiintegrin α5 subunit antibody (P1D6); α6, antiintegrin α6 subunit antibody (CLB701); αv, antiintegrin αv subunit antibody (P3G8); β1, anti-integrin β1 subunit antibody (6C6).

The precultured cells were transferred to a plate previously coated with βig-h3 protein and Hisβ-e and Hisβ-g prepared in Example 3 and further incubated at 37 ° C. for 1 hour before the cells attached to the substrate were hexosaminidase. Quantification by analysis of. The results of measuring the cell adhesion activity of βig-h3 are shown in FIG. 6A and the results of measuring the cell adhesion activity against Hisβ-e and Hisβ-g are shown in FIG. 6B . Each value represents the percentage of cells to attach in the absence of the monoclonal antibody, and each column represents the average of the results of three analyzes.

As can be seen in FIGS . 6A and 6B , cell adhesion on surfaces coated with βig-h3 recombinant protein was significantly inhibited by antibodies to α3 subunits and antibodies against β1 subunits but antibodies against other subunits. It was not inhibited by them. Although not shown, specific inhibition of cell adhesion mediated by βig-h3 by anti-integrin α3 and β1 antibodies was also observed in CHO cells.

Experimental Example 6 Analysis of Divalent Cation Sensitivity to Cell Adhesion Mediated by βig-h3

The effects of Mn ²⁺ , Mg ²⁺ and Ca ^{2+ on} the divalent cation sensitivity in cell adhesion mediated by βig-h3 were investigated.

CHO cells were suspended at 3 × 10 ⁵ cells / ml in 2 mM EDTA, 25 mM Hepes, 150 mM sodium chloride, pH 7.4 and incubated at 37 ° C. for 30 minutes. After incubation, cells were washed twice in Hepes buffered saline (150 mM sodium chloride, 25 mM hepes, pH 7.4) and resuspended in the same buffer.

Add an amount of cells to the microtiter bath and add Hepes buffered saline containing 2 times the final concentration of divalent cations (manganese chloride, magnesium chloride or calcium chloride) and Hepes buffered saline without divalent cations. Dispense ul by incubating each cell bath for 30 minutes at 37 ° C. under a humid atmosphere of 5% (v / v) carbon dioxide, and apply to Hisβ-b coated (10 μg / ml) dish for 1 hour. Further incubation. The number of cells attached to the substrate after the culture was quantified by analysis of hexosaminidase, and the results are shown in FIG. 7 .

As can be seen in FIG . 7 , cell adhesion to βig-h3 was strongly promoted by Mn ²⁺ and less by Mg ²⁺ , but not by Ca ²⁺ . These results suggest that cell adhesion to βig-h3 is mediated by integrin α3β1 and that the interaction is dependent on Mn ²⁺ and Mg ²⁺ .

Experimental Example 7 Correlation between Cell Attachment and Proliferation by βig-h3 and RGD Motif

Whether or not the RGD motif of βig-h3 is necessary for cell adhesion and proliferation activity was confirmed using various βig-h3 proteins having N- or C-terminal deletions prepared in Example 1.

CHO cells (2 × 10 ⁴ cells) were seeded on microtiter plates coated with 10 μg / ml BSA, pFN or 10 μg / ml other βig-h3 protein and incubated at 37 ° C. for 1 hour. Cells were rinsed and fixed, and then stained with crystal violet to quantify adhesion and diffusion of CHO cells to each substrate. The results are shown in Figs. 8A and 8B , and the values are the mean ± SD of the three measured values.

As shown in Figures 8a and 8b , all recombinant βig-h3 had almost the same effect on supporting cell adhesion and proliferation. As a result, it can be seen that βig-h3 supports cell adhesion and proliferation regardless of the RGD motif, and small deletion of the conserved domain does not affect its activity.

Experimental Example 8 SDS-PAGE and ND-PAGE Analysis of Recombinant Hisβ-e and Hisβ-g Proteins

Figure 9a shows the result of ND-PAGE analysis of Hisβ-e. BSA showing 66 kDa and 132 kDa was used as a molecular weight basis, and Hisβ-b was used for comparison. Lane 1 represents Hisβ-b, lane 2 represents Hisβ-e and lane 3 represents BSA. As can be seen from the results of FIG. 9A , Hisβ-e protein did not show multiple polymer bands in ND-PAGE. In addition, although the result was not shown, the same result as the above was obtained also in His (beta) -g.

9B shows the result of analysis by SDS-PAGE after crosslinking Hisβ-e with glutaraldehyde, and arrows indicate dimers. From the results of FIG. 9B , it can be seen that Hisβ-e mainly formed a dimer after crosslinking with glutaaldehyde. Although Hisβ-e showed oligomeric form at high concentrations of glutaraldehyde, the pattern was clearly different from Hisβ-a or Hisβ-b, which forms mostly high molecular weight cross-linked products at high concentrations of glutaraldehyde, and Hisβ- Most of e remained dimer. When the concentration of glutaraldehyde is increased, the intensity of the dimer band is further increased, indicating that Hisβ-e exists mainly as a dimer. In addition, although the result was not shown, the same result as the above was obtained also in His (beta) -g.

Experimental Example 9 Cell Attachment Ability of Hisβ-e and Hisβ-g

To determine whether Hisβ-e and Hisβ-g contain the ability to support cell adhesion and proliferation, experiments were carried out using BSA as a control and Hisβ-b as a control.

CHO cells (2 × 10 ⁴ ) were inoculated into 96 crude microtiter plates coated with Hisβ-e, Hisβ-b or BSA, and then incubated at 37 ° C. for 1 hour. After incubation, the degree of diffusion of CHO cells for each plate was confirmed by measuring absorbance at 405 nm, and the results are shown in FIG. 10A . In addition, the degree of diffusion of each plate was investigated by the area of diffusion, and the results are shown in FIG. 10B .

10A and 10B show that Hisβ-e can promote CHO cell adhesion and proliferation as strongly as Hisβ-b. Although the results are not shown, Hisβ-g also adheres to CHO cells the same as Hisβ-e. And could promote diffusion. This indicates that the nature of the polymerization and fibrous structure is not essential for βig-h3 to support cell adhesion and diffusion.

Next, CHO cells were preincubated for 30 min in a culture solution containing various concentrations of Hisβ-b or Hisβ-e in vitro, and then the cell suspension was transferred to Hisβ-e coated dishes and further cultured for 1 hour. After culturing to the determination of cell adhesion and proliferation showed the result in Fig. 10c, each value is the mean ± SD of 3 cultures. It can be seen from the above experiment that Hisβ-e inhibits cell adhesion in a positively dependent manner, and the cell adhesion and proliferation capacity of Hisβ-e are the same or slightly higher than Hisβ-b at the same concentration. Although the result was not shown, the same result was obtained also in His (beta) -g.

Experimental Example 10 Immobilization Analysis (Solid State Analysis)

In order to investigate the binding ability of the substrate proteins of various immobilized cells to βig-h3 used as the soluble ligand, the binding strength of Hisβ-b and Hisβ-e protein to the substrate protein of the immobilized cells was measured.

A 96-well Enzyme Linked Immunosorbent assay plate with a flat bottom was pre- overnight at 4 ° C. with 0.5 μg of various fixed extracellular molecules (ECM) proteins in 100 μl of a 20 mM carbonate buffer at pH 9.6. Coated. Extracellular matrix proteins used for coating were purified human plasma fibronectin, collagen types I and II of chicken (Chemicon International Inc. Temecula, CA, USA), bovine collagen types IV and VI (Chemicon), and mouse laminin (Chemicon). ) And bovine plasma albumin (Sigma).

Non-specific binding sites were blocked with phosphate buffered saline / 0.05% Tween 20 for 1 hour at 37 ° C., and 100 μl phosphate buffered saline / 0.05% Tween 20 was used at various concentrations of recombinant βig-h3 protein at 0.1-10 μg. Incubated at 37 ° C. for 2 hours. After incubation, each bath was washed with phosphate buffered saline / 0.05% Tween 20, incubated with anti-His-HRP antibody (Invitrogen) for 2 hours at 37 ° C, and washed again. To detect the interaction, 200 μl of a solution of o -phenylenediamine (Sigma) dissolved in 0.003% hydrogen peroxide solution and 1% methanol at a concentration of 0.1 mg / μl was added to each bath, and then at 37 ° C. in a dark place. Incubate for 1 hour. 50 μl of 3 M sulfuric acid was added to stop the reaction and the absorbance at 492 nm was measured with a Multiscan MCC / 340 microplate reader.

Exhibited a combination of Hisβ-b protein for other immobilized cell matrix protein in Figure 11a, it shows the binding of protein to the outer Hisβ-e the immobilized cell matrix protein in Figure 11b. Obviously high binding was observed for fibronectin in FIG. 11A , while moderate binding was observed for collagen types I, II, IV and VI and laminin. In contrast, as can be seen in Figure 11b Hisβ-e protein does not react at all with the extracellular matrix protein, it can be seen that one repeat domain is not sufficient for binding to the extracellular matrix protein.

Comparative Example 1 Adhesion and Diffusion Capability of Natural βig-h3 Isolated from Cells

As a comparative example, the cell adhesion and diffusion capacity of the original βig-h3 isolated from the cells were tested.

The Sma I / Bgl II DNA fragment of pBSβig-h3 was cloned into the EcoR V region of the pcDNA3.1 (-) / myc-His A (Invitrogen) vector to make pMycβ-a, which was Aoc I and Hind III of pMycβ-a. A PCR fragment corresponding to the C terminus was inserted at the site to prepare pMycβ-b, a mammalian expression vector of βig-h3. A diagram of a mammalian expression vector for full length βig-h3 is shown in FIG. 12 .

CHO cells were treated with α-MEM medium supplemented with 10% FBS (fetal bovine serum), 100 units / ml penicillin and 100 μg / ml streptomycin at 37 ° C. under 5% carbon dioxide (Minimum Essential Medium alpha, Gibco BRL Gaithersburg, MD, USA). In addition, HCE cells were supplemented with 15% FBS, 5 μg / ml insulin, 0.1 μg / ml choleratoxin and 10 ng / ml human epidermal growth factor (EGF). It was cultured in DMEM (Dubecco's Modified Eagle Medium) medium containing (DMEM / F-12, Gibco BRL). For transient expression, CHO cells and HCE cells were inoculated into 6-well culture plates at 2 x 10 5 cells / tan and 1.3 x 10 5 cells / tan, respectively, and 1 μg pMycβ-c DNA and 6 μl lipo for 5 hours at 37 ° C. It was transformed with lipofectamine ™ (Gibco BRL). Transformed cells were incubated for 48 hours in medium with 10% FBS before changing to medium without plasma.

The metastasized cells were further cultured for 48 hours to separate only the supernatant. Stably transformed CHO and HCE cells were selected using selection media containing 1.0 mg / ml or 2.0 mg / ml of G-418 (Sigma), respectively. Expression of βig-h3 in the culture supernatant was confirmed by immunoblotting using anti-βig-h3 antiserum, and the results are shown in FIGS. 13A and 13B , and each bar represents an average ± SD (n = 3). The expression of Mycβ-b, the full-length cDNA, was detected by anti-βig-h3 antiserum, not anti-Myc antibody because the C terminus was cleaved, wherein anti-βig-h3 antiserum was made for Hisβ-b protein.

As can be seen in FIGS . 13A and 13B , these stably metastasized HCE and CHO cells showed better adhesion and proliferation than untransferred HCE and CHO cells. From these results, it was found that βig-h3 isolated from HCE and CHO cells also supported their adhesion and diffusion. In addition, it was clear that the recombinant βig-h3 protein of the present invention, which was expressed in E. coli and isolated after being regenerated, was not significantly different from the original βig-h3 protein in terms of cell adhesion and proliferation.

According to the method of the present invention as described above, by using E. coli as a host, recombinant βig-h3 protein can be produced in large quantities, and recombinant βig-h3 produced in E. coli was purified and regenerated under denatured conditions, but the original form made in mammalian cells. It can support cell adhesion and diffusion in the same way as

Recombinant proteins comprising a second or fourth repeat domain of βig-h3 protein are water soluble, unlike full-length βig-h3 protein, easy to isolate and purify, and were not sufficient for interaction with other extracellular matrix proteins. Sufficient for cell adhesion and diffusion.

Therefore, the proteins of the present invention can be effectively used for inhibiting tumor formation and promoting wound healing.

Claims (8)

the second fas-1 domain (amino acid sequence corresponding to position 237-377 in SEQ ID NO: 2) or the fourth fas-1 domain (amino acid sequence corresponding to position 498-637 in SEQ ID NO: 2) of the βig-h3 protein alone Recombinant protein expressed with E. coli as a host, including one or more, or in combination. The recombinant protein of claim 1, wherein the recombinant protein supports cell adhesion and proliferation. Gene encoding the recombinant protein of claim 1. The method of claim 3, wherein the gene comprises at least one base sequence corresponding to position 711-1131 in SEQ ID NO: 1 or a base sequence corresponding to position 1494-1911 in SEQ ID NO: 1 or a mixture thereof gene. Recombinant expression vector pHisβ-g or pHisβ-e comprising the gene of claim 4. E. coli transformed with E. coli BL21 / Hisβ-g (Accession No .: KCTC 18010P) or E. coli BL21 / Hisβ-e (Accession No .: KCTC 18009P) transformed with the expression vector pHisβ-g or pHisβ-e ). 1) a cDNA fragment comprising one or more genes and selection markers comprising, alone or in combination, a nucleotide sequence encoding the second fas-1 domain of the βig-h3 protein or a nucleotide sequence encoding the fourth fas-1 domain; Manufacturing step; 2) preparing an expression vector into which the gene is inserted; 3) transforming Escherichia coli by transfection of the expression vector; 4) expressing the protein in the transformed Escherichia coli; And 5) Purifying the expressed protein using a selection label, the method of producing a recombinant protein of claim 1. The nucleotide sequence encoding the second fas-1 domain of step 1) is a nucleotide sequence corresponding to the position 711-1131 in SEQ ID NO: 1, and a nucleotide sequence encoding the fourth fas-1 domain. Is a nucleotide sequence corresponding to position 1494-1911 in SEQ ID NO: 1.