研究業績集

投稿論文2007年

タイトル

コロイダルリソグラフィーを使用して製造される抗体のナノパターン
Antibody Nanopatterns on Gold Fabricated by Colloidal Lithography

著者

シヴァシャンカ　クリシュナムティ, ミハエル　ヒンメルハウス
Sivashankar Krishnamoorthy, Michael Himmelhaus

雑誌名

平成19年度NPPP 実績報告書
装置利用　F産総 H19-129 p328-329

内容

Colloidal lithography has been combined with self-assembled monolayer (SAM) technology and reactive ion etching (RIE) to achieve nanopatterns of antibodies embedded in a protein-resistant matrix on a continuous gold film. The activity and specificity of such patterns in view of their antigen binding capacity was studied by means of surface plasmon resonance (SPR) and atomic force microscopy (AFM).

タイトル

Detection of prion protein immune complex for bovine spongiform encephalopathy diagnosis using fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

著者

藤井文彦1),堀内基広1),上野雅由2),坂田啓司1),長尾一生1),田村守1),金城政孝1)
1) 北海道大 2)富士レビオ株式会社

雑誌名

ANALYTICAL BIOCHEMISTRY 370 (2007) 131-141

内容

蛍光相関分光法(FCS)と蛍光相互相関分光法(FCCS)は、均一溶液や生細胞中の分子間相互作用を高感度測定するための優れた技術である。本研究において、我々はFCS,FCCSによるPrP測定法を開発した。FCCSのPrP検出限界はFCSより約3倍高く、PrPsc検出感度はELISAと同等であった。FCS,FCCSは均一状態におけるPrP免疫複合体をダイレクトに測定でき、かつ微量のサンプルと1回のみの攪拌操作で済むことから、BSE検査のオートメーション化に寄与するであろう。

タイトル

Glyceraldehyde-3-Phosphate Ferredoxin Oxidoreductase from Methanococcus Maripaludis

著者

Myong-Ok Park, Taeko Mizutani, and Patrik R. Jones

雑誌名

Journal of Bacteriology, Oct.2007, p.7281-7289

内容

The genome sequence of the non-sugar assimilating mesophile, Methanococcus maripaludis, contains three genes encoding enzymes (GAPN, GAPDH, GAPOR) potentially capable of catalyzing glyceraldehyde-3-phosphate (G3P) metabolism, including a homolog of Pyrococcus furiosus G3P:ferredoxin-oxidoreductase (mmGAPOR). GAPOR, whose homologs have been mainly found in archaea, catalyzes the reduction of ferredoxin coupled with oxidation of G3P. GAPOR has previously only been isolated and characterized from a sugar assimilating hyper-thermophile, Pyrococcus furiosus (pfGAPOR), and contains the rare metal tungsten as an irreplaceable cofactor. Active recombinant GAPOR was purified from Escherichia coli grown in minimal medium containing 100 mM sodium molybdate. In contrast, mmGAPOR obtained from cells grown in W-, W and Mo-containing media, and medium without added W and Mo did not display any activity. Activity and transcript analysis of putative G3P-metabolizing enzymes and corresponding genes was performed with M. maripaludis cultured under autotrophic conditions in chemically defined medium.
The activity of GAPOR was constitutive throughout the culture period and exceeded that of GAPDH by a factor of 10. As GAPDH activity only was detected in the gluconeogenic direction, whilst GAPN-activity was completely absent, only mmGAPOR catalyzes oxidation of G3P in M. maripaludis.
Recombinant mmGAPOR is post-transcriptionally regulated as it exhibits pronounced and irreversible substrate inhibition and is completely inhibited by 1 mM ATP. With support from flux balance analysis, it is concluded that the major physiological role of GAPOR in M. maripaludis most likely will be under non-optimal growth condition

タイトル

Current status and novel approaches for improving fermentative biomass-derived H2-production through genetic engineering of microbial metabolism

著者

Patrik R. Jones

雑誌名

Asian Biohydrogen Symposium Compendium Nov 9, 2007 p.2-15

内容

Microbial fermentation is a potentially promising method for renewable production of hydrogen. A major factor currently limiting commercial viability of hydrogen production is poor stoichiometric yield per mole of sugar. Although several approaches have been tried to enhance microbial hydrogen productivity, genetic engineering technologies for most promising microorganisms are either unavailable or severely limited.
Engineering of native H2-producing microorganisms is further limited by narrow or undefined substrate specificities and directionalities of most endogenousH2-pathways, setting an upper absolute limit to achievable H2-yield well below theoretical potential. An alternative approach instead is to choose an engineerable host, without regard for native H2-synthesis capabilities. Hydrogenases and pathways channelling electrons between carbon catabolism and hydrogenases therefore need to be introduced. However, mere introduction of new pathways is unlikely to be sufficient, as native host metabolism also will have to be engineered to favour the new pathways. A brief review of the current status of biological H2-production is presented, with a focus on fermentative processes. Selected recent advances are highlighted, including a summary of a research project following the abovementioned principles. The paper is concluded with a brief discussion on the potential role of computational modelling for advancing H2-production by genetic engineering.

タイトル

The Modular Organization of Domain Structures: Insights into Protein-Protein Binding.

著者

Antonio del Sol, Pablo Carbonell.

雑誌名

PLoS Comput Biol. 2007 Dec 7;3(12):e239.

内容

Domains are the building blocks of proteins and play a crucial role in protein-protein interactions. Here, we propose a new approach for the analysis and prediction of domain-domain interfaces. Our method, which relies on the representation of domains as residue-interacting networks, finds an optimal decomposition of domain structures into modules.
The resulting modules comprise highly cooperative residues, which exhibit few connections with other modules. We found that non-overlapping binding sites in a domain, involved in different domain-domain interactions, are generally contained in different modules. This observation indicates that our modular decomposition is able to separate protein domains into regions with specialized functions. Our results show that modules with high modularity values identify binding site regions, demonstrating the predictive character of modularity.
Furthermore, the combination of modularity with other characteristics, such as sequence conservation or surface patches, was found to improve our predictions. In an attempt to give a physical interpretation to the modular architecture of domains, we analyzed in detail six examples of protein domains with available experimental binding data. The modular configuration of the TEM1-beta-lactamase binding site illustrates the energetic independence of hotspots located in different modules and the cooperativity of those sited within the same modules.
The energetic and structural cooperativity between intramodular residues is also clearly shown in the example of the chymotrypsin inhibitor, where non-binding site residues have a synergistic effect on binding.
Interestingly, the binding site of the T cell receptor beta chain variable domain 2.1 is contained in one module, which includes structurally distant hot regions displaying positive cooperativity.
These findings support the idea that modules possess certain functional and energetic independence. A modular organization of binding sites confers robustness and flexibility to the performance of the functional activity, and facilitates the evolution of protein interactions.

タイトル

Engineering of a synthetic hydF-hydE-hydG-hydA operon for biohydrogen production

著者

M.Kalim Akhtar, Patrik R. Jones

雑誌名

ANALYTICAL BIOCHEMISTRY 373 (2008) 170-172

内容

Engineering microorganisms for enhanced H2-production requires introduction of new oxidoreductase:electron acceptor/donor:hydrogenase-pathways and multiple modifications to native host metabolism. As synthesis of active recombinant hydrogenases also is dependent upon coexpression of multiple maturation factors, we converged the individual hyd genes of Clostridium acetobutylicum in the form of a synthetic and extendable hydF-hydE-hydG-hydA operon. This framework is essential for introduction of the large number of heterolgous genes required to enhance biohydrogen production.

タイトル

Characterization of cap-shaped silver particles for surface-enhanced fluorescence effects.

著者

山口哲司,　彼谷高敏,　竹井弘之

雑誌名

Analytical Biochemistry　Vol.364, Issue2, 171-179 2007 2007年5月号

内容

表面増強蛍光効果を実現するために、ガラス基板へ高密度に吸着させた直径100nm微粒子単層へ銀を10nm蒸着し、ナノハーフシェルを作製した。蛍光分子を物理吸着もしくはサンドイッチ免疫反応系を介してナノハーフシェル上に配置し、共焦点蛍光スキャナーにて測定を行った。その結果、対照に比べて蛍光分子を物理吸着させた場合はシグナル値が約10倍上昇し、サンドイッチ免疫反応系においてはシグナル値が50倍以上と大幅な増加が認められた。

タイトル

Multifunctional Bio-Nano Patterns Derived from Colloidal Self-Assembly as Model Surfaces to Study Antigen-Antibody Interactions.

著者

S. Krishnamoorthy, J. P. Wright, O. Worsfold, T. Fujii, M. Himmelhaus

雑誌名

Technical Proceedings of the 2007 Nanotechnology Conference and Trade Show, Nanotech 2007 Vol.4, 590-593

内容

This paper presents a simple and effective means of preparing nanopatterns made of organic self-assembled monolayers on large surface areas by means of nanosphere lithography and exploring the capability of these patterns for immobilization of antibodies. The large pattern size allows for a detailed surface analysis via infrared spectroscopy and X-ray photoelectron spectroscopy, thereby revealing quality of the chemical patterns as well as allowing precise determination of the overall surface fraction of the functionalized patches. In a second step, the patterns are used for an in-situ surface plasmon resonance study on the physisorption of antibodies, followed by specific binding of antigens

タイトル

Modular architecture of protein structures and allosteric communications: potential implications for signaling proteins and regulatory linkages

著者

Del Sol A, Arauzo-Bravo MJ, Amoros D, Nussinov R

雑誌名

Genome biology 2007 May 25;8(5):R92

内容

Allosteric communications are vital for cellular signaling. Here we explore a relationship between protein architectural organization and shortcuts in signaling pathways. RESULTS: We show that protein domains consist of modules interconnected by residues that mediate signaling through the shortest pathways. These mediating residues tend to be located at the inter-modular boundaries, which are more rigid and display a larger number of long-range interactions than intra-modular regions. The inter-modular boundaries contain most of the residues centrally conserved in the protein fold, which may be crucial for information transfer between amino acids. Our approach to modular decomposition relies on a representation of protein structures as residue-interacting networks, and removal of the most central residue contacts, which are assumed to be crucial for allosteric communications. The modular decomposition of 100 multi-domain protein structures indicates that modules constitute the building blocks of domains. The analysis of 13 allosteric proteins revealed that modules characterize experimentally identified functional regions. Based on the study of an additional functionally annotated dataset of 115 proteins, we propose that high-modularity modules include functional sites and are the basic functional units. We provide examples (the Galphas subunit and P450 cytochromes) to illustrate that the modular architecture of active sites is linked to their functional specialization. CONCLUSION: Our method decomposes protein structures into modules, allowing the study of signal transmission between functional sites. A modular configuration might be advantageous: it allows signaling proteins to expand their regulatory linkages and may elicit a broader range of control mechanisms either via modular combinations or through modulation of inter-modular linkages.

タイトル

Ligand Binding and Circular Permutation Modify Residue Interaction. Network in DHFR.

著者

Hu Z, Bowen D, Southerland WM, Del Sol A, Pan Y, Nussinov R, Ma B.

雑誌名

PLoS Computational Biology. 2007 Jun 15;3(6):e117

内容

Residue interaction networks and loop motions are important for catalysis in dihydrofolate reductase (DHFR). Here, we investigate the effects of ligand binding and chain connectivity on network communication in DHFR. We carry out systematic network analysis and molecular dynamics simulations of the native DHFR and 19 of its circularly permuted variants by breaking the chain connections in ten folding element regions and in nine nonfolding element regions as observed by experiment. Our studies suggest that chain cleavage in folding element areas may deactivate DHFR due to large perturbations in the network properties near the active site. The protein active site is near or coincides with residues through which the shortest paths in the residue interaction network tend to go. Further, our network analysis reveals that ligand binding has "network-bridging effects" on the DHFR structure. Our results suggest that ligand binding leads to a modification, with most of the interaction networks now passing through the cofactor, shortening the average shortest path. Ligand binding at the active site has profound effects on the network centrality, especially the closeness.

タイトル

化学発光酵素免疫測定法を用いた献血者における高感度ヒトパルボウイルスB19抗原スクリーニング

著者

武田尋美1)、坂田秀勝1）、松林圭二1）、坂上尚仁2）、佐藤進一郎1）、伊原弘美1）、加藤俊明1）、池田久實1）
1)北海道赤十字血液センター　2)富士レビオ株式会社

雑誌名

日本輸血細胞治療学会誌　第53巻　第4号　467-472 2007年8月号

内容

ルミパルスPrestoを用いた高感度ヒトパルボウイルスB19抗原検出試薬（CLEIA-B19抗原）の開発を行った。B19 DNA陽性パネル（n=152）をCLEIA-B19抗原で測定した結果、RHA法より4Log高感度であることがわかった。また20プールNAT陰性の671検体をCLEIA-B19抗原で測定した結果、1例のみ偽陽性となった。以上のことよりCLEIA-B19抗原は感度特異性に優れ、献血者B19スクリーニングに有用であると考えられた。

タイトル

Electron microscopic estimation of removal of parvovirus B19(HPVB19) by nanofiltration with a novel filter membrane

著者

山口和人1)、高橋寛彦2)、宮崎　孔子3)、池田久實3)、宮川英二4)
1) 山口大学大学院医学系研究科生命実験医学分野生命科学実験施設
2) たかはし小児科医院
3) 北海道赤十字血液センター
4) 富士レビオ株式会社

雑誌名

Journal of Membrane Science 298 (2007) 99-109

内容

クロマトグラフィー精製した感染性有する高濃度ヒトパルボウイルスB19（感染価：109～1013TCID50/mL）を用いて15、20、35nmのフォローファイバーでのウイルスの除去をろ過前後のウイルス感染価（KU812Ep6細胞使用）、PCR法及びろ過後のフォローファイバー断面の電子顕微鏡観察により検証した。ヒトパルボウイルスB19は15nmフォローファイバーで完全に除去出来ることが確認された。