• Molecules and Cells
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• 제45권7호
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• pp.444-453
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• 2022

Multivalent macromolecular interactions underlie dynamic regulation of diverse biological processes in ever-changing cellular states. These interactions often involve binding of multiple proteins to a linear lattice including intrinsically disordered proteins and the chromosomal DNA with many repeating recognition motifs. Quantitative understanding of such multivalent interactions on a linear lattice is crucial for exploring their unique regulatory potentials in the cellular processes. In this review, the distinctive molecular features of the linear lattice system are first discussed with a particular focus on the overlapping nature of potential protein binding sites within a lattice. Then, we introduce two general quantitative frameworks, combinatorial and conditional probability models, dealing with the overlap problem and relating the binding parameters to the experimentally measurable properties of the linear lattice-protein interactions. To this end, we present two specific examples where the quantitative models have been applied and further extended to provide biological insights into specific cellular processes. In the first case, the conditional probability model was extended to highlight the significant impact of nonspecific binding of transcription factors to the chromosomal DNA on gene-specific transcriptional activities. The second case presents the recently developed combinatorial models to unravel the complex organization of target protein binding sites within an intrinsically disordered region (IDR) of a nucleoporin. In particular, these models have suggested a unique function of IDRs as a molecular switch coupling distinct cellular processes. The quantitative models reviewed here are envisioned to further advance for dissection and functional studies of more complex systems including phase-separated biomolecular condensates.

• Bulletin of the Korean Chemical Society
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• 제30권5호
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• pp.1101-1106
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• 2009

Multivalent and multi-specific antibodies can provide valuable tools for bio-medical research, diagnosis and therapy. In antigen-antibody interactions, the avidity of antibodies depends on the affinity and the number of binding sites.$^1$ As artificial multivalent antibody agents, single chain Fv-streptavidin fusion tetramer proteins $(scFv-SA)_4$ have been previously tested.$^{1,\;2}$ Although, the Fab domain is known to be more stable than scFv in animal models,$^{3,\;4}$ it has never been used to make a multivalent agent with a streptavidin fusion. In this study, we prepared tetra-valent $(Fab-cSA)_4$ by fusing Fab with core streptavidin (cSA). This molecule was made using inclusion body production, refolding and chromatography purification. Affinities of the Fab-cSA tetramer and a scFv-cSA tetramer to a cell surface antigen were compared by ELISA using biotin-HRP. The Fab-cSA tetramer showed higher binding avidity than the scFv-cSA tetramer. The higher binding avidity of the Fab-cSA tetramer demonstrates its potential as a therapeutic agent for target-specific antibody therapy.

• 한국고분자학회:학술대회논문집
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• 한국고분자학회 2006년도 IUPAC International Symposium on Advanced Polymers for Emerging Technologies
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• pp.265-265
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• 2006

Multivalent interactions, which are characterized by the simultaneous binding of multiple ligands on multiple receptors, are prevalent in biological system. We have shown that it is able to make a supramolecular aggregate coated with multiple functional molecules fairly easily by simply mixing one building block. In this particular example, a mannose-coated object was able to agglutinate bacterial cells with cognate binding partners through multivalent interactions. This kind of strategy can be applied in developing materials that can selectively remove pathogens. Supramolecular assembly of this type should be very useful in exploring multivalent biological interactions.

• Molecules and Cells
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• 제43권11호
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• pp.899-908
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• 2020

Intrinsically disordered proteins or regions (IDPs or IDRs) are widespread in the eukaryotic proteome. Although lacking stable three-dimensional structures in the free forms, IDRs perform critical functions in various cellular processes. Accordingly, mutations and altered expression of IDRs are associated with many pathological conditions. Hence, it is of great importance to understand at the molecular level how IDRs interact with their binding partners. In particular, discovering the unique interaction features of IDRs originating from their dynamic nature may reveal uncharted regulatory mechanisms of specific biological processes. Here we discuss the mechanisms of the macromolecular interactions mediated by IDRs and present the relevant cellular processes including transcription, cell cycle progression, signaling, and nucleocytoplasmic transport. Of special interest is the multivalent binding nature of IDRs driving assembly of multicomponent macromolecular complexes. Integrating the previous theoretical and experimental investigations, we suggest that such IDR-driven multiprotein complexes can function as versatile allosteric switches to process diverse cellular signals. Finally, we discuss the future challenges and potential medical applications of the IDR research.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권6호
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• pp.460-464
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• 2000

Extracellular polymeric substances (EPS) are believed to play a role in the binding and formation of microbial flocs. However, the precise role is not well known. Sludge settling characteristics and the carbohydrate to protein ratio in EPS were tested with various airflow rates in this study. Sludge was collected from three modified sequencing batch reactors (SBRs), which were operated at 16$\^{C}$ with an airflow rate of 0.8L/min, 3L/min and 6L/min, respectively. During the operation, the reactor operated at an airflow rate of 0.8L/min showed sludge volume index (SVI) of 80 to 90ml/g and a constant ratio of carbohydrate to protein in the EPS, while a significant increase in the SVI was seen in the other reactors. Sludge bulking increased the amount of carbohydrate in the EPS, while kept protein almost constant in the airflow rate of 3L/min ad 6L/min. Surface charge also increased with increases in the carbohydrate to protein ratio in the EPS, which weakens the attraction between the EPS and multivalent cations. The ratio of carbohydrate to protein in the EPS was tween the EPS and multivalent cations. The ratio of carbohydrate to protein in the EPS was inferred to be essential for bioflocculation.

• Journal of Microbiology and Biotechnology
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• 제20권4호
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• pp.789-797
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• 2010

The limited information on differential gene expression in the different serotypes of Actinobacillus pleuropneumoniae has significantly hampered the research on the pathogenic mechanisms of this organism and the development of multivalent vaccines against A. pleuropneumoniae infection. To compare the gene expressions in the A. pleuropneumoniae strains CVCC259 (serotype 1) and CVCC261 (serotype 3), we screened the differentially expressed genes in the two strains by performing representational difference analysis (RDA). Northern blot analyses were used to confirm the results of RDA. We identified 22 differentially expressed genes in the CVCC259 strain and 20 differentially expressed genes in the CVCC261 strain, and these genes were classified into 11 groups: (1) genes encoding APX toxins; (2) genes encoding transferrin-binding protein; (3) genes involved in lipopolysaccharide (LPS) biosynthesis; (4) genes encoding autotransporter adhesin; (5) genes involved in metabolism; (6) genes involved in the ATP-binding cassette (ABC) transporter system; (7) genes encoding molecular chaperones; (8) genes involved in bacterial transcription and nucleic acid metabolism; (9) a gene encoding protease; (10) genes encoding lipoprotein/membrane protein; and (11) genes encoding various hypothetical proteins. This is the first report on the systematic application of RDA for the analysis of differential gene expression in A. pleuropneumoniae serotypes 1 and 3. The determination of these differentially expressed genes will serve as an indicator for future research on the pathogenic mechanisms of A. pleuropneumoniae and the development of a multivalent vaccine against A. pleuropneumoniae infection.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권4호
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• pp.293-300
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• 2001

A polyrotaxane-biotin conjugate was synthesized and its interaction with streptavidin measured using surface plasmon resonance(SPR) detection. A biodegradable polyrotaxane in which ca, 22 molecules of ${\alpha}$-cyclodextrina(${\alpha}$-CDs) were threaded onto a poly(ethylene oxide) chain(M$\sub$n:4,000) capped with benzyloxycarbonyl-L-phenylalanine was conjugated with a biotin hydorazide and 2-aminoethanol after activing the hydroxyl groups of ${\alpha}$-CDs in the polyrotaxane using N, N'-carbonyldiimidazole. The results of the high-resolution $^1$H-nyclear lmagnetic resonance($^1$H-NMR)spectra and gel permeation chromatography of the conjugate showed that ca, 11 biotin molecules were actually introduced to the polyrotaxane scaffold. An SPR analysis showed that the binding curves of the biotin molecules in the conjugate on the streptavidin-deposited surface changed in a concentration dependent manner, indicating that the biotin in the conjugate was ac-tually recognized by streptavidin. The association equilibrium constant(K$\sub$a/) of the interaction be-tween the conjugate and steptavidin tetramer was of the order 10$\^$7/. These results suggest that polyrotaxane is useful for scaffolds as a polymeric ligand in biomedical fields.

• 미생물학회지
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• 제54권4호
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• pp.330-342
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• 2018

Streptavidin (STR)과 Biotin system은 Biotin의 Streptavidin에 대한 높은 비공유 친화력(non-covalent affinity; $K_D=10^{-14}M$)과 4 Biotin 결합부위를 갖는 Streptavidin의 tetramer 구조로 인해 복수의 항원결합부위 및 복수의 항원특이성을 갖는 항체를 제조할 수 있기 때문에 가장 활발하게 연구되고 있다. 이 system을 활용하기 위해 우리는 Streptomyces avidinii 염색체 DNA로부터 PCR을 통해 Streptavidin (STR) 유전자를 증폭하고 이를 TRAIL (tumor necrosis factor ${\alpha}$ related apoptosis induced ligand) receptor인 death receptor 4 (DR4)에 특이적으로 결합하는 hAY4 single-chain Fv 항체유전자에 융합시켰다. 대장균에서 발현시킨 STR에 융합된 hAY4 ScFv (hAY4-STR) 항체는 가열시킨 SDS-PAGE에서 43 kDa monomer를 나타내었다. 그러나 가열하지 않은 SDS-PAGE와 Size-exclusion chromatography에서는 tetramer인 172 kDa을 나타내었는데 이는 hAY4 ScFv-STR 항체가 STR의 자연적인 비공유결합에 의해 유도된 tetramer를 형성하고 있음을 나타내고 있다. 본 융합 단백질은 Ouchterlony assay와 ELISA에서 보여주는 것처럼 자연 Streptavidin과 유사한 Biotin 결합력을 유지하고 있었다. ELISA와 Westernblot을 이용하여 정제된 hAY4-STR 융합항체의 DR4 항원결합력 또한 확인하였다. 게다가 표면 플라즈몬 공명(surface plasmon resonance) 분석에서 hAY4 ScFv-STR tetramer는 tetramerization에 의해 hAY4 ScFv monomer보다 60배 더 높은 항원결합력을 나타내었다. 요약하면 hAY4 ScFv-STR 융합단백질은 E. coli에서 soluble tetramer로 성공적으로 발현 및 정제되었으며 Biotin과 DR4 항원에 동시에 결합함을 보여 주었다. 이는 bifunctional and tetrameric ScFv 항체를 제조 할 수 있음을 제시해 주고 있다.

• 대한소아치과학회지
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• 제35권1호
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• pp.73-82
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• 2008

치아우식은 주로 mutans streptococci에 의해 야기되는 감염성 질환으로서 주 원인균에는 streptococcus mutans가 있다. S. mutans가 치아우식을 유발하는 분자 생물학적 기전은 몇 가지 단계를 포함한다. 먼저 S. mutans는 AgI/II와 같은 세포 표면의 섬유성 단백질을 매개로 치면의 타액성 피막에 일차적으로 부착한다. 두번째 단계에서 자당의 존재하에 glucosyltransferase(GTF)는 glucan과 같은 다당체를 합성하게 된다. 마지막으로 이렇게 합성된 glucan은 glucan binding proteins와 상호작용하여 치면세균막을 형성해서 세균의 군집화를 가능하게 한다. 많은 실험과 임상연구에서 S. mutans의 주요 항원(Ag I/II, GTFs, GBPs)들이 치아우식 병리기전에 영향을 준다고 알려져 왔고, 따라서 이런 항원들이 면역계에 작용하여 치아우식을 막는 백신으로 이용 가능하다. 본 실험은 streptococcus mutans GS-5로부터, GTFb, GTFc, GTFd 유전자를 복제하고 염기서열분석을 하였으며, 이중 GTFd가 먼저 재조합 단백질 생산을 위해 발현 벡터에 클로닝 되었으며, 이로부터 단백질이 발현됨을 확인하였다. 이번 실험에서 얻은 순수 GTF 항원은 동물실험을 통해 특정 GTF 활성부위에 대한 항체 생산에 이용될 수 있을 것이다.