使用表面電漿共振儀研究藍色鏈黴菌末端蛋白和粒連結蛋白的交互作用

鏈黴菌屬的革蘭氏陽性細菌，有三分之二所生產的抗生素，運用在人類或獸醫的用藥之中。末端蛋白基因（Tpg）需要複製特定的鏈黴菌染色體或質體的線性分子，和粒連結蛋白 (Tap) 中染色體和質體的線性形式是息息相關的。在這項研究中，利用了波長的訊號製作了特殊的表面共振電漿（SPR），經由４個不同的通道，去觀察鏈黴菌中Tap與Tpg的相互作用。我們把含有histidine結構的蛋白質表現在大腸桿菌中，在此稱為TapC與TpgC，須先由離子交換層析法去純化與透析，再去執行 SPR。然後在進行所有的SPR實驗之前，我們須先改質金屬表面，使表面接上甲基，再經由Jung et al. 所製作的數學公式，和停留在金屬表面的蛋白，去看出兩者的交互作用。

關鍵字

鏈黴菌；藍色鏈黴菌末端蛋白；表面電漿共振儀；藍色鏈黴菌；蛋白的交互作用；粒連結蛋白

並列摘要

The genus Streptomyces is a gram-positive soil dwelling bacterium that currently produces over two-thirds of the naturally derived antibiotics in human and veterinary medicine. Streptomyces is unusual among bacteria for having protein-capped linear plasmids and chromosomes. The terminal protein gene (Tpg) is required for the specific replication of Streptomyces chromosomes or plasmids as linear molecules, and the telomere associated protein (Tap) is essential for the propagation of Streptomyces chromosomes and plasmids in linear form. In this work, a custom-built surface plasmon resonance (SPR) sensor based on wavelength interrogation with a four-channel Teflon flow cell was used to investigate the interaction between Tpg and Tap from the specie Streptomyces coelicolor, which is genetically the best known representative of the genus Streptomyces. The target proteins, herein called TpgC and TapC, were initially His-tagged and expressed in Escherichia coli (E. coli). The bacterial cultures were purified using immobilized metal ion affinity chromatography (IMAC) and dialyzed before running in the SPR sensor. A methyl-terminated self-assembled monolayer (SAM) immobilized on the sensor substrate was used in all SPR experiments. The amount of proteins immobilized on the sensor surface was computed based on the quantitative mathematical formalism established by Jung et al.

並列關鍵字

surface plasmon resonance ； Telomere associated protein ； Terminal protein gene ； Streptomyces ； Streptomyces coelicolor ； protein interaction

參考文獻

1. Bentley, S. D., K. F. Chater, et al. (2002) “Complete Genome Sequence of the Model Actinomycete Streptomyces Coelicolor A3(2).” Nature 417: 141 - 147.

3. Bao, K. and S. N. Cohen (2001). "Terminal Proteins Essential for the Replication of Linear Plasmids and Chromosomes in Streptomyces." Genes & Development 15: 1518 – 1527.

4. Yang, C. C., C.-H. Huang, et al. (2002). “The Terminal Proteins of Linear Streptomyces Chromosomes and Plasmids: A Novel Class of Replication Priming Proteins.” Molecular Microbiology. 43(2): 297 – 305.

5. Bao, K. and S. N. Cohen (2003). “Recruitment of Terminal Protein to the Ends of Streptomyces Linear Plasmids and Chromosomes by a Novel Telomere-Binding Protein Essential for Linear DNA Replication.” Genes & Development. 17: 774 – 785.

6. Chen, C. W. (2007). Microbial Linear Plasmids. Microbiology Monographs. F. Meinhardt and R. Klassen. Berlin Heidelberg, Springer-Verlag 7.

國際替代計量

使用表面電漿共振儀研究藍色鏈黴菌末端蛋白和粒連結蛋白的交互作用

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