• Journal of Microbiology and Biotechnology
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• v.25 no.1
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• pp.119-126
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• 2015

Among the various human growth factors, epidermal growth factor (hEGF, consisting of 53 amino acids) has various effects on cell regeneration, stimulation of proliferation, migration of keratinocytes, formation of granulation tissues, and stimulation of fibroblast motility, which are important for wound healing. Owing to their multiple activities, EGFs are used as pharmaceutical and cosmetic agents. However, their low productivity, limited target specificity, and short half-life inhibit their application as therapeutic agents. To overcome these obstacles, we fused the collagen-binding domain (CBD) of Vibrio mimicus metalloprotease to EGF protein. About 18 or 12 amino acids (aa) (of the 33 total amino acids), which were essential for collagen-binding activity, were combined with the N- and C-termini of EGF. We constructed, expressed, and purified EGF (53 aa)-CBD (18 aa), EGF (53 aa)-CBD (12 aa), CBD (18 aa)-EGF (53 aa), and CBD (12 aa)-EGF (53 aa). These purified recombinant proteins increased the numbers of cells in treated specimens compared with non-treated specimens and control hEGF samples. The collagen-binding activities were also evaluated. Furthermore, CBD-hybridized hEGF induced phosphorylation of the EGF receptor. These results suggested that these fusion proteins could be applicable as small therapeutic agents in wound tissue healing.

• Journal of Periodontal and Implant Science
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• v.38 no.4
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• pp.621-628
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• 2008

Purpose: Osteopontin is one of the major non-collagenous protein of hard tissue. Use of peptide domain of biologically active protein has some advantages. The objective of this experimental study is evaluation of periodontal regenerative potency of synthetic peptide gel which containing collagen binding domain of osteopontin in the degree III periodontal defect of beagle dogs. Material and Methods: Experimental degree III furcation defect was made in the mandibular third and fourth premolar of beagles. Regenerative material was applied during flap operation. 8 weeks after regenerative surgery, all animals were sacrificed and histomorphometric measurement was performed to calculate the linear percentage of the new cementum formation and the volume percentage of new bone formation. Result: The linear percent of new cementum formation was 41.6% at control group and 67.1% at test group and there was statistically significant difference. The volume percent of new bone formation was 52.1% at control group and 58.9% at test group. Conclusion: As the results of present experiment, synthetic peptide gel containing collagen binding domain of osteopontin significantly increase new bone and cementum formation in the degree III furcation defect of canine mandible.

• Journal of Life Science
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• v.23 no.3
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• pp.347-354
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• 2013

Innate immunity is the ability to differentiate infectious agents from self. The innate immune system is comprised of a complicated network of recognition and effector molecules that act together to protect the host in the early stage of an infectious challenge. Mannose-binding lectin (MBL or mannose-binding protein, MBP) belongs to the family of $Ca^{2+}$-dependent lectins (C-type lectin with a collagen-like domain), which are considered an important component of innate immunity. While it is associated with increased risk and severity of infections and autoimmunity, the most frequent immuno-deficiency syndrome was reported to be low MBL level in blood. Deficiency of human MBL is caused by mutations in the coding region of the MBL gene. Rat homologue gene of human MBL gene was used to study functions of wild type and mutant MBL proteins. Although extensive studies have yielded the structural information of MBL, the functions of MBL, especially mutant MBL, still require investigation. We previously reported the cloning of rat wild-type MBL gene and the production of a truncated form of MBL protein and its antibody. Here, we present the cloning of mutant MBL cDNA in collagen-like domain (R40C, G42D, and G45E) using site-directed mutagenesis and differential behaviors of wild type and mutant MBL in cells. The major difference between wild type and mutant MBL was that while wild type MBL was secreted, mutant MBL was inhibited for secretion, retained in endoplasmic reticulum, and still functioned as a lectin.

• Journal of Life Science
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• v.8 no.3
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• pp.235-240
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• 1998

Matrix metalloproteinases(MMPs) are a group of zinc enzymes responsible for degradation of the matrix components such as collagen and proteoglycans in normal embryogenesis and remodeling and in many disease processes such as arthritis, cancer, periodontitis, and osteprocess. Genetically distince MMPs have been characterized and their genes have been cloned thus far from a variaty of species but not from fishes. In this stydy, a mmp cDNA was cloned by using RT-PCR(reverse transcriptase dependent polymerase chain reaction) from Scylliorhinus toraxzame(shark), agroup of cartilaginous fish, abundant in the coast of Pusan, Korea. It has 74% base homologue with membrane type matrix matalloproteinase-3 genes(mt3-mmps) from human, rat and chick, and also shows more than 90% residue homologue with them. In addition, it has cysteine switch domain, zinc binding domain(HExGH motif), propeptide cleavage site, and RRKR motif, which are present in MMPs. This result indicates that cDNA fragment cloned here may be mt3-mmp or its analogous gejne cDNA fragment of Scylliorhinus torzame.

• BMB Reports
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• v.40 no.4
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• pp.532-538
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• 2007

Mouse ficolin A is a plasma protein with lectin activity, and plays a role in host defense by binding carbohydrates, especially GlcNAc, on microorganisms. The ficolin A subunit consists of an N-terminal signal peptide, a collagen-like domain, and a C-terminal fibrinogen-like domain. In this study, we show that ficolin A can be synthesized and oligomerized in a cell and secreted into culture medium. We also identify a functionally relevant signal peptide of ficolin A by using MS/MS analysis to determine the N-terminal sequence of secreted ficolin A. When the signal peptide of mouse ficolin A was fused with enhanced green fluorescent protein (EGFP), EGFP was released into HEK 293 cell medium, suggesting that the signal peptide can efficiently direct ficolin A secretion. Moreover, our results suggest that the signal peptide of ficolin A has potential application for the production of useful secretory proteins.

• Journal of Life Science
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• v.19 no.2
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• pp.284-288
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• 2009

The innate immune system is important for the first line of host defence against infectious agents, which have penetrated the mechanical barriers. Mannose-binding lectin (MBL or mannan-binding protein, MBP) is a serum protein that is synthesized in the liver as a part of the acute phase response. MBL binds to carbohydrate structures presented by a wide range of pathogenic bacteria, viruses, fungi, and parasites. MBL is synthesized as a monomer that has a carboxy-terminal carbohydrate recognition domain, a neck region and a collagen region. Low MBL level was reported to be the most frequent immuno-deficiency syndrome. Although extensive studies have yielded detailed information on the structure of MBL, functions of the MBL complex are not fully understood yet. We, here, present cloning process of MBL cDNA from the rat liver and production of truncated recombinant MBL protein using a bacterial expression system in order to produce anti-MBL polyclonal antibody. Anti-MBL polyclonal antibody was raised in a New Zealand rabbit and its affinity was tested against recombinant protein using western blot technique. MBL cDNA, recombinant protein and anti-MBL antibody could be used as great arsenals to dissect cellular biochemistry of MBL.

• Journal of Periodontal and Implant Science
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• v.41 no.6
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• pp.293-301
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• 2011

Purpose: We have previously reported that tetra-cell adhesion molecule (T-CAM) markedly enhanced the differentiation of osteoblast-like cells grown on anorganic bone mineral (ABM). T-CAM comprises recombinant peptides containing the Arg- Gly-Asp (RGD) sequence in the tenth type III domain, Pro-His-Ser-Arg-Asn (PHSRN) sequence in the ninth type III domain of fibronectin (FN), and the Glu-Pro-Asp-Ilu-Met (EPDIM) and Tyr-His (YH) sequence in the fourth fas-1 domain of ${\beta}$ig-h3. Therefore, the purpose of this study was to evaluate the cellular activity of osteoblast-like cells and the new bone formation on ABM coated with T-CAM, while comparing the results with those of synthetic cell binding peptide (PepGen P-15). Methods: To analyze the cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed, andto analyze gene expression, northernblot was performed. Mineral nodule formations were evaluated using alizarin red stain. The new bone formations of each group were evaluated using histologic observation and histomorphometrc analysis. Results: Expression of alkaline phosphatase mRNA was similar in all groups on days 10 and 20. The highest expression of osteopontin mRNA was observed in the group cultured with ABM/P-15, followed by those with ABM/T-CAM and ABM on days 20 and 30. Little difference was seen in the level of expression of collagen type I mRNA on the ABM, ABM/T-CAM, and ABM/P-15 cultured on day 20. There were similar growth and proliferation patterns for the ABM/T-CAM and ABM/P-15. The halo of red stain consistent with $Ca^{2+}$ deposition was wider and denser around ABM/T-CAM and ABM/P-15 particles than around the ABM particles. The ABM/T-CAM group seemed to have bone forming bioactivity similar to that of ABM/P-15. A complete bony bridge was seen in two thirds of the defects in the ABM/T-CAM and ABM/P-15 groups. Conclusions: ABM/T-CAM, which seemed to have bone forming bioactivity similar to ABM/P-15, was considered to serve as effective tissue-engineered bone graft material.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.81-89
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• 2004

Tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and lymphotoxin-$\alpha$ (LT-$\alpha$, TNF-$\beta$) can initiate and perpetuate human diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and insulin-dependent diabetes mellitus (IDDM). TNFs can be blocked by the use of soluble TNF receptors. However, since monomeric soluble receptors generally exhibit low affinity or function as agonists, the use of monomeric soluble receptors has been limited in the case of cytokines such as TNF-$\alpha$, TNF-$\alpha$, interleukin (IL)-1, IL-4, IL-6, and IL-13, which have adapted to a multi component receptor system. For these reasons, very high-affinity inhibitors were created for the purpose of a TNFs antagonist to bind the TNFR and trigger cellular signal by using the multistep polymerase chain reaction method. First, recombinant simple TNFR-Ig fusion proteins were constructed from the cDNA sequences encoding the extracellular domain of the human p55 TNFR (CD120a) and the human p75 TNFR (CD120b), which were linked to hinge and constant regions of human $IgG_1$ heavy chain, respectively using complementary primers (CP) encoding the complementary sequences. Then, concatameric TNFR-Ig fusion proteins were constructed using recombinant PCR and a complementary primer base of recombinant simple TNFR-Ig fusion proteins. For high level expression of recombinant fusion proteins, Chinese hamster ovary (CHO) cells were used with a retroviral expression system. The transfected cells produced the simple concatameric TNFR-Ig fusion proteins capable of binding TNF and inactivating it. These soluble versions of simple concantameric TNFR-Ig fusion proteins gave rise to multiple forms such as simple dimers and concatameric homodimers. Simple TNFR-1g fusion proteins were shown to have much more reduced TNF inhibitory activity than concatameric TNFR-Ig fusion proteins. Concatameric TNFR-Ig fusion proteins showed higher affinity than simple TNFR-Ig fusion proteins in a receptor inhibitor binding assay (RIBA). Additionally, concatameric TNFR-Ig fusion proteins were shown to have a progressive effect as a TNF inhibitor compared to the simple TNFR-Ig fusion proteins and conventional TNFR-Fc in cytotoxicity assays, and showed the same results for collagen induced arthritis (CIA) in mice in vivo.