![]() The designed DNA backbone consists of six ODNs, of which two have a length of 26 nt and four have a length of 55 nt. For our purposes, we designed a DNA backbone composed of five solid segments, each connected by a flexible linker ( Fig. The DNA scaffolds in these works are rigid and proteins on the scaffold cannot associate with each other. Recently, enzymes have been aligned on a DNA scaffold to facilitate consecutive reactions –. DNA is widely used to construct nano-architecture by taking advantage of its specific recognition of complementary nucleotide sequences. We used DNA structure as a backbone for the designed alignment of several proteins. sfGFP-ODN was eluted by the low salt buffer supplemented with 400 mM imidazole. The column was washed with the low salt buffer and removal of unreacted N 3-ODN was monitored by absorbance of at 280 nm. The eluted solution was applied to a Ni-column (Ni-sepharose, GE healthcare) to remove the unreacted N 3-ODN. The column was washed with a low-salt buffer (20 mM Tris-HCl, pH7.4, 100 mM NaCl) and sfGFP-ODN was eluted by a high-salt buffer (20 mM Tris-HCl, pH7.4, 500 mM NaCl). sfGFP-ODN has negative charges due to the phosphate backbone of DNA and has higher affinity to the anion exchange column than does free protein. To remove remaining free protein, the reaction mixture was applied to an anion exchange column (DEAE-650M TOYOPEARL). Yield of the sfGFP-ODN production was analyzed by SDS-PAGE. The reaction mixture containing 20 µM His 6-sfGFP-Cys, 20 µM DBCO-PEG 4-Maleimide (Click Chemistry Tools, USA), 40 µM N 3-ODN in buffer (20 mM Tris-HCl, pH7.4, and 100 mM NaCl) was incubated at 37☌ for 10 hours. SfGFP-ODN Preparation by Strain-promoted Azide-alkyne Catalyst-free Click Chemistry The supernatant was applied to anion exchange column (DEAE-650M TOYOPEARL), eluted by step-wise increase of NaCl concentration (0–100 mM), and subsequently purified by Ni-sepharose column (GE healthcare). In the case of sfGFP purification, cell lysate was heat-treated at 80☌ for 15 min before centrifugation. Harvested cells were sonicated in buffer (20 mM Tris-HCl, pH 7.4, 1 mM DTT, a Complete protease inhibitor mixture tablet (Roche Applied Science)) and centrifuged. Proteins were expressed in Escherichia coli BL21(DE3) at 20☌ for 20 hr. A206K mutations (monomer propensity) and S208F/V224L mutations (dimer propensity) were also introduced. A cysteine residue was introduced at the N-terminus of CFP (Cys-CFP) and at the 173 position of YFP (Cys-YFP), taking into consideration their orientations in the dimer. We used Cerulean and Venus as improved CFP and YFP. This increases local concentrations of the proteins and will enable association of proteins with very weak interactions.Įxpression plasmid pET21c-His 6-sfGFP-Cys was generated from pET21c-wild-type GFP by site-directed mutagenesis using the PrimeSTAR mutagenesis basal kit (Takara, Japan). Here we report a procedure to align several different proteins, all connected to a single flexible DNA backbone. For effective formation of complex, proteins should be linked with high flexibility enough to allow proteins to move and turn freely to associate with each other. Fused protein has structural restriction and is often difficult to express in a soluble form. Chemical cross-linking has a limited reactivity and is not easy to control the number of cross-linked proteins. However, these methods have some limitations. To study such complex, component proteins of the complex are chemically cross-linked or genetically fused as a single polypeptide to prevent the complex from dissociation. In many cases, however, interactions are transient or weak, producing a complex that is difficult to isolate. ![]() When the interactions are strong, proteins associate into a stable homo- or hetero-oligomer complex that can be isolated relatively easily. Protein-protein interactions play a critical role in numerous biological processes, and understanding the nature of each interaction is of central importance in biology and biotechnology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. Y.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (No. Received: JAccepted: NovemPublished: December 26, 2012Ĭopyright: © 2012 Nojima et al. Citation: Nojima T, Konno H, Kodera N, Seio K, Taguchi H, Yoshida M (2012) Nano-Scale Alignment of Proteins on a Flexible DNA Backbone. ![]()
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