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The effects of angiogenesis inhibitor from the extract libraries of Korean and Chinese medicinal plants were investigated using a protein microarray chip. Protein chip was constructed by immobilization of integrin α5β1 on protein chip base plates and employed for screening active extracts that inhibit the integrin-fibronectin interaction from the extract libraries. The 100 extracts of medicinal plants were obtained from extract bank of National Institute of Crop Science, RDA. The 14 extracts among 100 extract libraries were shown efficient inhibition activity for the interaction between integrin-fibronectin. The medicinal plants of 14 extracts were Vitex negundo var. incisa (Lam.) C.B. Clarke, Epimedium koreanum Nakai, Cedrela sinensis A. Juss, Ipomea aquatica Forsk, Schisandra chinensis Baill, Pulsatilla koreana Nakai, Paeonia lactiflora Pall. var.hortensis Makino, Oenothera odorata, Allium chinense, Allium victorialis var. platyphyllum MAKINO, Polygonatum odoratum Druce var. pluriflorum Ohwi, Hosta lancifolia, Agrimonia pilosa L. var. japonica Nakai and Potentilla chinensis SER. The Paeonia lactiflora, Oenothera odorata, and Agrimonia pilosa from these 14 extracts libraries were shown strong inhibition activity of integrin α5β1.

나노기술과 바이오기술의 융합연구에 의해 나노바이오기술이 발전되고 있다. 나노바이오기술의 중요한 응용연구 중의 하나로서, 진단이나 바이오센서 분야에서 단백질-단백질 및 단백질-바이오물질간의 상호작용을 연구하기 위한 단백질 센서 칩이 개발되어 왔다. 본 논문에서는 단백질의 선택적 고정화를 위한 새로운 생체고분자 기질로 PHA를 이용하는 첫 번째 예로서, 단백질-단백질 및 항원-항체 반응의 구현을 나타내고자 하였다. 본 시스템은 PHA 표면 위에서 PHA depolymerase의 SBD와의 선택적 결합에 기반한 것으로, PHA depolymerase의 SBD와 융합된 단백질이 PHA가 코팅된 표면 위에 spotting 될 수 있고 미세접촉인쇄방법에 의해 PHA 위에 미세패턴이 제조되어지는 것을 알 수 있었다(52, 53). 이러한 새로운 전략이 PHA depolymerase의 SBD와 다른 단백질을 융합함으로서 미세 spotting과 미세패터닝이 가능하게 되었고 항원-항체의 생물학적 반응을 통해 많은 바이오센서 칩 연구에 응용될 수 있음을 확인하였다. 또한, PHA 마이크로비드에도 PHA depolymerase의 SBD와 융합된 단백질을 고정시킴으로서 항원-항체 반응을 유도할 수 있음을 확인하였다(54). PHA의 구조를 변경하여 PHA 기판, PHA 필름, PHA 미세패턴, PHA 마이크로 비드 등을 이용할 수 있으며 multiplex assay를 동시에 진행할 수 있는 다양한 융합 단백질을 사용할 수 있을 것이다. 생분해성 플라스틱으로서 성공적으로 개발된 PHA를 이용한 새로운 플랫폼 기술이 PHA depolymerase의 SBD를 이용함으로서 특이적이고 선택적인 단백질의 고정화에 이용될 수 있음을 확인하였다. 본 전략이 다양한 단백질-단백질 및 단백질-바이오물질반응을 이용한 바이오칩 및 바이오센서의 응용연구에 유용하게 사용될 것이다.

Polyhydroxyalkanoates (PHAs) are a family of microbial polyesters that can be produced by fermentation from renewable resources. PHAs can be used as completely biodegradable plastics or elastomers. In this paper, novel applications of PHAs in biosensor are described. A general platform technology was developed by using the substrate binding domain (SBD) of PHA depolymerase as a fusion partner to immobilize proteins of interest on PHA surface. It could be shown that the proteins fused to the SBD of PHA depolymerase could be specifically immobilized onto PHA film, PHA microbead, and microcontact printed PHA surface. We review the results obtained for monitoring the specific interaction between the SBD and PHA by using enhanced green fluorescent protein, red fluorescent protein, single chain antibody against hepatitis B virus preS2 surface protein and severe acute respiratory syndrome coronavirus surface antigen as model proteins. Thus, this system can be efficiently used for studying protein-protein and possibly protein-biomolecule interactions for various biotechnological applications.

현재 많은 대학과 기업에서 다양한 방법으로 상용화가 가능한 protein microarray의 개발을 위해 많은 연구를 집중하고 있다. Protein microarray의 제작 및 분석 조건을 최적화하기 위한 연구도 진행되고 있지만 protein microarray로부터 얻은 분석 결과를 모든 연구자들이 공유하고 통합하기 위한 노력이 절실한 실정이다. 뿐만 아니라, PCR 같은 무한 확장 방법이 존재하지 않는 단백질의 특성을 고려할 때, 좀 더 실용적인 protein microarray를 많이 만들기 위해 서는 수많은 단백질들과 결합할 수 있는 특이성이 높고 결합력이 강한 capture molecule들을 개발하는 것이 필수이다. 그러나 이러한 장애에도 불구하고 protein microarray는 아주 적은 시료량으로 high-throughput assay가 가능하다는 장점 때문에 현재의 생명과학의 발전 추세로 볼 때 앞으로 protein microarray가 조만간 실용화될 것이며 이의 시장성은 매우 클 것으로 기대된다. 보다 빠른 실용화를 위해 서는 protein microarray의 개발에 필요한 기반 기술의 개발과 동시에 이를 활용하기 위한 contents의 개발도 절실히 요구된다.

Analysis of protein interactions/functions in a microarray format has been of great potential in drug discovery, diagnostics, and cell biology, because it is amenable to large-scale and high-throughput biological assays in a rapid and economical way. In recent years, the protein microarray have broaden their utility towards the global analysis of protein interactions on a proteome scale, the functional activity analysis based on protein interactions and post-translational modifications (PTMs), and the discovery of biomarkers through profiling of protein expression between sample and reference pool. As a promising tool for proteomics, the protein microarray technology has advanced outstandingly over the past decade in terms of surface chemistry, acquisition of relevant proteins on a proteomic level, and detection methods. In this article, we briefly describe various techniques for development of protein microarray, and introduce developmental state of protein microarray and its applications.

We describe a simple and rapid chemical strategy for preparing carbohydrate microarrays and utilize these arrays for the characterization of carbohydrate-protein interactions. Carbohydrate microarrays were prepared by reductive amination-mediated immobilization of amine-modified carbohydrate onto NHS-activated glass slide. The carbohydrate conjugates were confirmed by MALDI-TOF MS and 1H NMR. Carbohydrate microarray presenting six disaccharide were evaluated by profiling the binding specificities of several lectins. The results showed that the array selectively bound the corresponding lectins with relative binding affinities. We identified lectin and carbohydrate detection limit concentration on this array using WGA(Wheat Germ Agglutinin) as lectin and N,N'-diacetylchitobiose(GlcNAcβ-12Man) as carbohydrate. We also evaluated interactions between cholera toxin B subunit and GM1, GM1 analogues such as asialo GM1, GM3 on this array. As a result, GM1 pentasaccharide showed a strong affinity and specificity in interaction, but asialo GM1 and GM3, which is lacking of terminal Neu5Ac and galactose-N-acetylgalactosamine, respectively, showed a low affinity. We proposed that this carbohydrate microarray system could be applicable for feasible studies, like sensing of pathogenic bacterium or detection of toxin protein.

Carbohydrate-protein interactions are involved in various biological events, including viral and bacterial infection, immune response, differentiation and development, the progression of tumor cell metastasis. Carbohydrate microarrays are new technology that has potentiated the high-throughput analysis of carbohydrate-protein interaction and contributes to significant advances of glycomics. Here, we proposed a facile, efficient, and cost-effective method whereby diverse carbohydrate types are modified in a single step and directly immobilized onto a glass surface, with retention of functional orientation. We modified various types of carbohydrates by reductive amination, in which reducing sugar groups were coupled with the linker. The modified carbohydrates were covalently attached to an amine-reactive NHS-activated glass surface by formation of stable amide bonds. This method was applied for construction of functional carbohydrate microarray platform to analyze carbohydrate-protein interactions. This array by using our method can be powerful tool for screening the specificity of GBPs (glycan-binding proteins) such as lectins, growth factors, antibody, and microbial toxins, and identification of carbohydrate-protein interactions.

Carbohydrate-protein interactions are involved in various biological events, including viral and bacterial infection, immune response, differentiation and development, the progression of tumor cell metastasis. Carbohydrate microarrays are new technology that has potentiated the high-throughput analysis of carbohydrate-protein interaction and contributes to significant advances of glycomics. Here, we proposed a facile, efficient, and cost-effective method whereby diverse carbohydrate types are modified in a single step and directly immobilized onto a glass surface, with retention of functional orientation. We modified various types of carbohydrates by reductive amination, in which reducing sugar groups were coupled with the linker. The modified carbohydrates were covalently attached to an aminereactive NHS-activated glass surface by formation of stable amide bonds. This method was applied for construction of functional carbohydrate microarray platform to analyze carbohydrate-protein interactions. This array by using our method can be powerful tool for screening the specificity of GBPs (glycan-binding proteins) such as lectins, growth factors, antibody, and microbial toxins, and identification of carbohydrateprotein interactions

Cell cycle-dependent protein kinases (CDKs) regulates the cells entry into mitosis during the transition from G2 to M phase, by phosphorylating a group of key proteins including RNA polymerase II and histone H1. A recombinant substrate for the enzyme was expressed as a recombinant protein from a plasmid pMFS-cdc2 in E. coli, and it was purified. In this study, we present a strategy for the detection of a phosphorylation of a substrate catalysed by cdc2 protein kinase by using a radioisotope detection technique in microarrays. We conclude that the use of Mep45-fused substrate in developing a protein biochip for the detection of the activity of cdc2 protein kinase by using a RI detection technique is highly economical and can be used for both high throughput screening and studying protein-protein interactions. Keywords: Cdc2 protein kinase, Gene cloning, Microarrays, Radioisotope detection

이 연구에서는 법랑모세포 분화과정에서 APin의 기능을 알아보고자 APin-protein microarray를 시행한 후 치아발생과 관련이 있는 MEF2, Aurora kinase A, BMPR-IB와 EF-hand calcium binding protein을 분석하여 다음과 같은 결과를 얻었다. 1 CMV-APin construct를 transfection하여 APin의 과발현을 유도한 경우에는 MEF2와 Aurora kinase A 둘 모두에서 발현이 현저히 감소한 반면에, APin의 발현억제를 유도한 경우에는 둘 모두 변화가 없었다. 2. APin의 과발현을 유도한 경우에는 BMPR-IB와 EF-hand calcium binding protein 모두에서 발현이 크게 증가한 반면, APin을 발현억제 시킨 경우에는 BMPR-IB는 변화가 없었고, EF-hand calcium binding protein은 현저히 감소하였다. 위의 결과들로 보아 APin 단백질은 MEF2, Aurora kinase A, BMPR-IB, EF-hand calcium binding protein과 상호작용하여 법랑모세포의 분화와 석회화 과정 중에 중요한 역할을 하는 것으로 사료된다.

Protein microarray or protein chips is potentially powerful tools for analysis of protein-protein interactions. APin cDNA was previously identified and cloned from a rat odontoblast cDNA library. The purpose of this study was to investigate the APin-protein interactions during ameloblast differentiation. Protein microarray was carried with recombinant APin protein and MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein were selected among 74 interacting proteins. Immortalized ameloblast cells (ALCs) were transfected with pCMV-APin construct and U6-APin siRNA construct. After transfection, the expression of the mRNAs for four proteins selected by protein micoarrays were assessed by RT-PCR. The results were as follows: 1. APin expression was increased and decreased markedly after its over-expression and inactivation, respectively. 2. Over-expression of the APin in the ALCs markedly down-regulated the expression of MEF2 and Aurora kinase A, whereas their expression remained unchanged by its inactivation. 3. Expression of BMPR-IB and EF-hand calcium binding protein were markedly increased by the over-expression of the APin in the ALCs, whereas expression of BMPR-IB remained unchanged and expression of EF-hand calcium binding protein was markedly decreased by its inactivation. These results suggest that APin plays an important role in ameloblast differentiation and mineralization by regulating the expression of MEF2, Aurora kinase A, BMPR-IB and EF-hand calcium binding protein.

Background: Empirical evidences for efficacy of hot spring (HS) water in inflammatory skin disorders have not been substantiated with sufficient, immunological “hard evidence”. Mageumsan HS water, characterized by its weakly-alkaline properties and low total dissolved solids content, has been known to alleviate various immune-inflammatory skin diseases, including atopic dermatitis (AD). Objective: The trial attempted to quantitatively analyze in vitro expression levels of chemical mediators in cutaneous inflammation from HaCaT cell line treated with Mageumsan HS, and suggest the likely mode of action through which it exerts the apparent anti-inflammatory effects in AD. Methods: Using membranebased human antibody array kit, customized to include 30 different, keratinocyte-derived mediator proteins, their expression levels (including interleukin [IL]-1, IL-6, IL-8, thymic stromal lymphopoietin, thymus and activation-regulated chemokine, and granulocyte macrophage colony-stimulating factor) were assessed in vitro. Selected key proteins were further quantified with enzyme-linked immunosorbent assay. Results: There was a clear pattern of overall suppression of the mediators, especially those noted for their pro-inflammatory role in AD (monocyte chemoattractant protein [MCP]-1, regulated on activation, normal T cell expressed and secreted, cutaneous T-cell-attracting chemokine, Eotaxin, and macrophage inflammatory protein-1α, etc.). Also, reduced expression of involucrin and cytokeratin 1 was also reduced in the HS-treated group. Conclusion: The present study has shown that Mageumsan HS water may exert its effects on inflammatory skin disorders through regulation of proinflammatory cytokines. These evidences are to be supported with further future investigations to elucidate immunological mechanism behind these beneficial effects of HS water in the chronically inflamed skin of AD.

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (>326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.

Binding sequence specificity of the Arabidopsis transcription factor AtMYB77 was determined by the quadruple 9-mer-based protein binding microarray (Q9-PBM) analysis. The position weight matrix and Wilcoxon-Mann-Whitney test with total 1843 clustered signals revealed that the full size AtMYB77 protein binds specifically to the consensus sequence pAACnG, where p represents T or C; n, A, G, C or T. This sequence is known as the type I binding site for MYB transcription factors. This result indicates that functional diversity among the type I MYB transcription factors is not due to their binding specificity on a particular promoter sequence.

A protein binding microarray (PBM) analysis was performed to determine the nucleotide sequence responsible for the specific binding of FUS3, a transcription factor in Arabidopsis that is considered to be a master regulator of seed development. Full-length FUS3 cDNA was fused in-frame with sequences encoding the fluorescent protein DsRed and a polyhistidine tag, and expressed in Escherichia coli. The partially purified fusion protein was incubated with the quadruple 9-mer PBM. An analysis of fluorescent images revealed that the fusion protein recognized and bound to the consensus sequence CATGCA. This result suggests that, among the nucleotides contained within the RY motif (CATGCA[TG]), six nucleotides (CATGCA) are sufficient for FUS3-specific binding. It appears that an additional 'TG' at the 3' end of the RY motif found in seed-specific gene promoters was not necessary for binding specificity. Notably, FUS3 may also bind to the palindromic RY motif in its dimeric form.

In this paper, we have been described a new constructing method of multichannel biosensor using self-assembly by magnetic force interaction. A metal particle and an array was fabricated by photolithographic. Biomaterials were immobilized on the metal particle. The array and the particles were mixed in a buffer solution, and were arranged by magnetic force interaction and self-assembly. A quarter of total Ni dots were covered by the particles. The binding direction of the particles was controllable, and condition of particles was almost with Au surface on top. The particles were successfully arranged on the array. The biomaterial activities were detected by chemiluminescence.

Background : Insulin-like growth factor binding protein 3 (IGFBP3), a member of six proteins with a high affinity for insulin-like growth factors (IGFs), seems to modulate the effects of IGFs on cells and to regulate cell proliferation through the IGF-independent pathway. We assessed the role of IGFBP3 in the colorectal carcinoma detected by cDNA microarray. Methods : To identify molecular alterations in the colorectal carcinoma, we analyzed gene expression profiles of the colorectal adenocarcinoma by means of a cDNA microarray representing 7,500 genes. Of the differentially expressed genes, the author assessed the insulinlike growth factor binding protein 3 (IGFBP3) gene at the protein level using immunohistochemistry. Results : The expressions of 21 and 16 genes were noted to have more than fivefold increases or decreases in the colonic adenocarcinoma tissue compared with the noncancerous colonic mucosal tissue. The differentially expressed genes include those associated with cell proliferation/apoptosis, signal transduction/transcription, metabolizing enzymes, cytoskeleton, angiogenesis, ion channel, extracellular matrix and others. Of the total 68 cases of colorectal adenocarcinomas observed, 34 cases (50%) showed positive immunohistochemical stainings for IGFBP3. Conclusion : In this study, it is suggested that IGFBP3 plays a role in colorectal carcinogenesis. And combining an immunohistochemistry with a cDNA microarray can facillitate the rapid characterization of a candidate novel molecular target.