• KSBB Journal
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• 제13권2호
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• pp.125-132
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• 1998

Protein affinity membrane was prepared via the coating of chitosan gel on the porous flat polysulfone membrane surface, followed by the immobilization f the reactive dye (Cibacron Blue 3GA) to the chitonsan gel. The maximum protein binding capacity of affinity membrane was about 70${\mu}g/cm^2$ determined by the batch adsorption experiments of human serum albumin (HSA). Using module of this membrane, the characteristics of protein chromatography were investigated through the experiments of elution and frontal chromatography of HSA. This membrane module promises as a chromatography column, since it represented a lower pressure drop and a greater reproducibility. The protein separation ratio was significantly influenced by the flow rate of mobile phase and the injection quantity of HSA. The dynamic protein binding capacity of module decreased from the equilibrium binding capacity with increasing flow rate and approached the value of 15 - 20 ${\mu}g/cm^2$ for flow rates above 6 mL/min.

• 대한의생명과학회지
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• 제7권2호
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• pp.59-64
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• 2001

Nebulin is an unusually large actin-binding protein specific to the skeletal muscle of vertebrates. The correlation of nebulin size with thin filament length have led to the suggestion that nebulin acts as a molecular ruler for the length of thin filaments. An SH3 domain occupies the C terminus of nebulin, in the sarcomeric Z-disk and is preceded by a 120-residue stretch containing multiple putative phosphorylation sites. SH3 domain mediates protein-protein interaction involved in the subcellular localization of proteins, cytoskeletal organization and signal transduction. However the binding partner and physiological role of nebulin SH3 domains remains unknown. Using the yeast two-hybrid system, we identified supervillin, an actin-binding protein, as a nebulin SH3 domain-interacting protein. The SH3 domain of nebulin binds to the sequence encoding amino acids 977 to 1335 of supervillin. But the sequence encoding amino acids 977 to 1335 displays weaker binding than the sequence encoding amino acids 977 to 1788.

• BMB Reports
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• 제36권4호
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• pp.344-348
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• 2003

Protein kinase CKII is composed of two catalytic ($\alpha$ or $\alpha$') subunits and two regulatory ($\beta$) subunits. The $CKII{\beta}$ subunit is thought to mediate the tetramer formation and interact with other target proteins. However, its physiological function remains obscure. In this study, point mutants of $CKII{\beta}$ that are defective for the L41 binding were isolated by using the reverse two-hybrid system. A sequence analysis of the point mutants revealed that Asp-26, Met-52, and Met-78 of $CKII{\beta}$ are critical for L41 binding; Asn-67 (and/or Lys-139) and Met-52 are important for $CKII{\beta}$ homodimerization. Two point mutants, R75 and R83, of $CKII{\beta}$ interacted with L5, topoisomerase $II{\beta}$, and CKBBP1/SAG, but not with the wild-type $CKII{\beta}$. This indicates that $CKII{\beta}$ homodimerization is not a prerequisite for its binding to target proteins. These $CKII{\beta}$ point mutants may be useful in exploring the biochemical physiological functions of $CKII{\beta}$.

• 생명과학회지
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• 제8권4호
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• pp.400-409
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• 1998

Formation of adduct was studied in benzo(a)pyrene(BP)- and doxorubicin(Dx)-treated human NC-37 cells and isolated nuclei. Major adducts formed were determined by fluorescence absorption spectrophotometery and DNA-lin-ked protein assay. When isolated nuclei were exposed to carcinogens BP and DMBA, and anticancer drugs m-AMSA, ellipticine and Dx, varying degrees of adduct formation occured between DNA-protein complex and these drugs. When the mixture was centrifuged 1.7 M sucrose solution, binding BP and DMBA appeared to be similar between the sediment and the supernatant. When the sediment was centrifuged again with 0.35% polymin-P, the amount of BP bound was 2-fold greater in the protein(1077$\pm$55cpm) than in DNA fraction (470$\pm$20cpm), whereas that of DMBA was 1.6-fold greater in the DNA than in protein fraction. In the case of m-AMSA, ellipticine and Dx, the amount of binding was slightly greater in supernatant than in sediment in centrifugation with 1.7 M sucrose, and more than 3 times greater in the DNA- than in protein- fraction in centrifugation with 0.35% polymin P. DNA fractions which associated with a subset of nonhistone chromosomal protein were isolated from NC-37 cells exposed to $^{3}$H-BP and $^{14}$C-Dx. They were separated into two distince components DNA-S and DNA-P by centrifugation with 2M Nacl chromatin extraction. The results indicated that the amount of $^{3}$H-BP bound was 6.0-fold greater in DNA-P as compared with DNA-S, while that of $^{14}$C-Dx binding appreaed to be 6.2-fold greater in DNA-S than in DNA-P fraction. When $^{3}$H-BP binding wasdetermined in the presence of cold Dx, the amount of binding was reduced only in the DNA-P fraction, indicating that the interaction between DNA and protein is decreased. Gene expression by these drugs, BP treated cells were increased to compare with nomal cells but reduced by treatment with BP-Dx. These results suggest that the protein moiety which tightly bound to DNA-P fraction may play an important role in the regulation of gene expression.

• 한국식품저장유통학회지
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• 제22권2호
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• pp.297-302
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• 2015

저활용 단백질로부터 칼슘 결합물질을 분리하기 위해 돼지 육골분과 진주담치 단백질을 단백질 분해 효소인 alcalase를 이용하여 가수분해물을 제조하였고, 체내 흡수가 용이한 3 kDa 이하로 한외여과 하였다. 돼지 육골분 가수분해물은 Mono Q 컬럼을 통해 분리하였고, 진주담치 가수분해물의 경우 Q-Sepharose로 분리 하여 각각 2개, 3개의 peptide fraction을 얻어 각 fraction의 칼슘 결합력을 측정하였다. 그 결과 MBM F2와 Mussel F3에서 가장 높은 칼슘 결합력을 나타내었고, 따라서 본 연구 결과로 얻어진 가수분해물들은 칼슘 보충 소재로 활용될 수 있다고 판단된다.

• BMB Reports
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• 제29권2호
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• pp.175-179
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• 1996

Viral surface proteins are known to play an essential role in attachment of the virus particle to the host cell membrane. In case of the hepatitis B virus (HBV) several reports have described potential receptors on the target cell side, but no definite receptor protein has been isolated yet. As for the viral side, it has been suggested that the preS region of the envelope protein, especially the preS1 region, is involved in binding of HBV to the host cell. In this study, preS1 region was recombinantly expressed in the form of a maltose binding protein (MBP) fusion protein and used to identify and visualize the expression of putative HBV receptor(s) on the host cell. Using laser scanned confocal microscopy and by FACS analysis, MBP-preS1 proteins were shown to bind to the human hepatoma cell line HepG2 in a receptor-ligand specific manner. The binding kinetic of MBP-preS1 to its cellular receptor was shown to be temperature and time dependent. In cells permeabilized with Triton X-100 and treated with the fusion protein, a specific staining of the nuclear membrane could be observed. To determine the precise location of the receptor binding site within the preS1 region, several short overlapping peptides from this region were synthesized and used in a competition assay. In this way the receptor binding epitope in preS1 was revealed to be amino acid residues 27 to 51, which is in agreement with previous reports. These results confirm the significance of the preS1 region in virus attachment in general, and suggest an internalization pathway mediated by direct attachment of the viral particle to the target cell membrane.

• 미생물학회지
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• 제55권3호
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• pp.181-190
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• 2019

대장균 세포분열 조절에 관여하는 DicA 단백질은 $37^{\circ}C$보다 $25^{\circ}C$에서 DNA에 더욱 잘 결합한다. 그러나 DicA 단백질의 온도의존적 DNA 결합에 대한 분자적 원인은 명확하지 않다. 본 연구에서는 DicA 단백질이 어떻게 DNA에 결합하며, 왜 온도 의존적 결합양상을 보이는지 알아보았다. In vivo DNA 결합 분석 결과 RovA나 SlyA와 같은 DicA의 상동성 단백질과는 달리 DicA는 N 말단에 있는 DNA 결합 도메인을 이용하여 20개의 염기쌍으로 이루어진 dicC 조절자 유전자(Oc)에 결합함을 보여주었다. 또한 in vivo 실험에서 DicA는 $37^{\circ}C$ 보다 $25^{\circ}C$에서 DNA에 더 잘 결합하는 것으로 알려진 Cnu 또는 H-NS의 영향을 받지 않고 자체적으로 Oc에서의 온도 의존적 DNA 결합을 보인다. 하지만 정제된 DicA 단백질을 이용한 in vitro binding 실험에서는 온도 의존적 DNA 결합이 관찰되지 않았다. Crude 단백질을 이용한 실험에서 DicA 단백질의 온도 의존적 DNA 결합이 관찰되는 것으로 보아 DicA의 온도 의존적 DNA (Oc) 결합은 crude 단백질내에 존재하는 아직 알려지지 않은 in vivo factor에 의해 일어난다.

• BMB Reports
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• 제39권3호
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• pp.319-328
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• 2006

SecA protein, a cytoplasmic ATPase, plays a central role in the secretion of signal peptide-containing proteins. Here, we examined effects of signal peptide and ATP on the oligomerization, conformational change, and membrane binding of SecA. The wild-type (WT) signal peptide from the ribose-binding protein inhibited ATP binding to soluble SecA and stimulated release of ATP already bound to the protein. The signal peptide enhanced the oligomerization of soluble SecA, while ATP induced dissociation of SecA oligomer. Analysis of SecA unfolding with urea or heat revealed that the WT signal peptide induces an open conformation of soluble SecA, while ATP increased the compactness of SecA. We further obtained evidences that the signal peptide-induced oligomerization and the formation of open structure enhance the membrane binding of SecA, whereas ATP inhibits the interaction of soluble SecA with membranes. On the other hand, the complex of membrane-bound SecA and signal peptide was shown to resume nucleotide-binding activity. From these results, we propose that the translocation components affect the degree of oligomerization of soluble SecA, thereby modulating the membrane binding of SecA in early translocation pathway. A possible sequential interaction of SecA with signal peptide, ATP, and cytoplasmic membrane is discussed.

• 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.

• 한국잠사곤충학회지
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• 제30권1호
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• pp.25-32
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• 1988

누에 유충에 있어서 체액 JH 결합단자질(JHBP)의 생리적 역할을 알기 위하여, 체액 중의 JH 1과 결합하는 단자질을 polyacrylamide gel 전기영동으로 분리하고, 또 발육에 따른 JH 결합단자질의 활성 변화를 측정한 결과는 다음과 같다. 1. 누에 체액중에서 JH 1과 결합하는 단자질은 JH 결합지질단자질(JH-LP)과 Jh 결합단자질(JHBP) 등 JH결합단자질의 Rm은 0.81∼0.33이었고, JH 결합단자질의 Rm은 0.81∼0.84이었다. 두 단자질의 Rm은 성별, 발육시기에 관계없이 일정하였다. 2. 체액단자질의 함량은 발육의 진행에 따라 점차 증가하여 토사기에 가장 높았으며 전용기에 다소 감소하였고 암컷의 함량은 숫컷보다 높았다. 3. 체액 ml 당 JH 결합단자질의 활성은 영의 초기에 낮았고 토사기에 최대로 나타난 후 전용기에 다소 감소하였다. 4. 체액 ml 당 JH 결합단자질의 활성 변화는 체액단자질의 함량의 변화양상과 매우 유사하였다. 5. 체액단자질 mg 당 JH 결합단자질의 활성은 영의 초기에 높았고 5령 6일에서 전용기까지 낮은 수준을 유지하였다.