Enzyme-based protein tagging system

Various protein-labeling methods based on the specific interactions between genetically encoded tags and synthetic probes have been proposed to complement fluorescent protein-based labeling. In particular, labeling methods based on enzyme reactions have been intensively developed by taking advantage of the highly specific interactions between enzymes and their substrates. In this approach, the peptides or proteins are genetically attached to the target proteins as a tag, and the various labels are then incorporated into the tags by enzyme reactions with the substrates carrying those labels. On the other hand, we have been developing an enzyme-based protein-labeling system distinct from the existing ones. In our system, the substrate protein is attached to the target proteins as a tag, and the labels are incorporated into the tag by post-translational modification with an enzyme carrying those labels followed by tight complexation between the enzyme and the substrate protein. In this review, I summarize the enzyme-based protein-labeling systems with a focus on several typical methods and then describe our labeling system based on tight complexation between the enzyme and the substrate protein

Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane

The nuclear envelope (NE) is a double membrane that segregates nuclear components from the cytoplasm in eukaryotic cells. It is well-known that the NE undergoes a breakdown and reformation during mitosis in animal cells. However, the detailed mechanisms of the NE dynamics are not yet fully understood. Here, we propose a method for the fluorescent labeling of the NE in living cells, which enables the tracing of the NE dynamics during cell division under physiological conditions. In our method, labeling of the NE is accomplished by fixing green fluorescent protein carrying the nuclear localization signal on the inner nuclear membrane based on a unique biotinylation reaction from the archaeon Sulfolobus tokodaii. With this method, we observed HeLa cells during mitosis by confocal laser scanning microscopy and succeeded in clearly visualizing the difference in the timing of the formation of the NE and the nuclear lamina

Efficacy of the Pulse Pressure Generator during Cardiopulmonary Bypass Training Using the Extracorporeal Circulation Simulator

Objective: Cardiopulmonary bypass during cardiac surgery is an essential procedure, and the perfusionist needs to have sufficient education and training. Simulation training is suitable in such cases. We have developed a simulation system for cardiopulmonary bypass training (extracorporeal circulation simulator [ECCSIM]) and reported its efficacy. ECCSIM had no pulse pressure generator, so some perfusionists have mentioned that the operational feeling during training differs from that in real clinical cases. In this study, we have developed a new pulse pressure generator and examined the efficacy of this system during cardiopulmonary bypass training using ECCSIM. Materials and Methods: Results were observed as wave patterns during simulation of extracorporeal circulation with and without pulse pressure flow. Operational feeling during training of extracorporeal circulation was compared using a questionnaire survey, based on the Japanese version of the NASA Task Load Index (NASA-TLX), a subjective index, completed by seven perfusionists. Results: With the addition of pulse pressure flow, fluctuation of arterial flow at low speed with the centrifugal pump increased, and operation of extracorporeal circulation became unstable. The questionnaire survey, including ‘Operational feeling of centrifugal pump’, ‘Feeling of afterload’, and ‘Display of pressure monitor’, showed results similar to that of the clinical situation using pulse pressure flow. The difficulty of simulator operation in extracorporeal circulation was significantly greater in the group with pulse pressure flow. Mental/physical load examined with NASA-TLX increased with pulse pressure flow. Conclusion: Using a new pulse pressure generator with ECCSIM was effective in extracorporeal circulation training

Combination of Myogenic and Neurogenic Motor Evoked Potential Monitoring During Thoracoabdominal Aortic Surgery

A 64-year-old woman was evaluated for thoracoabdominal aortic aneurysms (TAAAs). Preoperative computed tomography showed a TAAA extending from the level of the diaphragm to the renal arteries. The Adamkiewicz artery (AKA) arose at the Th10 level, close to the aneurysm, and an abdominal aortic prosthesis and left iliac artery aneurysm were detected. Myogenic and neurogenic motor evoked potentials (MEPs) were monitored during the surgical repair of the TAAA, and there were differences between the two types of MEPs during surgery. Both MEPs fell below 50% of their baseline levels during surgery, which suggested critical ischemia, but the decrease in the myogenic MEP occurred at a different time from the decrease in the neurogenic MEP. A time-course analysis suggested that AKA reimplantation was unnecessary and all intercostal arteries were ligated. Both MEPs recovered completely by the end of surgery and there were no postoperative neurologic deficits. Our findings suggest that the combination of myogenic and neurogenic MEP monitoring is helpful in evaluating spinal cord injury during the surgical repair of TAAAs

Stepwise Preparation of a Polymer Comprising Protein Building Blocks on a Solid Support for Immunosensing Platform

In immunosensing, immobilization of the antibody on the sensing platform significantly influences the performance of the sensor. Herein, we propose a novel antibody-immobilization method based on a protein-polymer chain containing multiple copies of an antibody-binding protein, the Z-domain. In our approach, the Z-domain-containing polymer is prepared on the surface of the sensing platform with a biotinylation reaction from the archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property by which biotin protein ligase (BPL) forms an extremely stable complex with its biotinylated substrate protein (BCCP). Here, we employed two types of engineered proteins: one was the fusion protein of BCCP with the Z-domain (BZB), in which BCCP was genetically attached to the N- and C-termini of the Z-domain; the other was a BPL dimer prepared by connecting two BPL molecules with a cross-linking reagent. We applied these two engineered proteins alternately onto the BPL-modified solid support of the surface plasmon resonance sensor chip, and succeeded in growing polymer chains comprising multiple units of BZB and the BPL dimer. The antibody-binding capability of the Z-domain-containing polymer thus prepared is adjustable by controlling the number of cycles of protein addition and the surface density of the polymer on the solid support

金属配位官能基を有するDNA配位子の合成と機能評価

第1章 序論 第2章 金属配位官能基を有するアントラキノン誘導体とDNAの相互作用 第3章 金属配位官能基を有するオリゴヌクレオチドとDNAの相互作用 第4章 オリゴヌクレオチドとランタノイド金属イオンの複合体の発光挙動 第5章 結論Made available in DSpace on 2012-07-10T07:18:42Z (GMT). No. of bitstreams: 3 sueda1.pdf: 12846648 bytes, checksum: bf2a3ea9320ec3a86c843ea0b38bce9d (MD5) sueda2.pdf: 9031604 bytes, checksum: 1a4cb590eb05b224828c43609d267709 (MD5) sueda3.pdf: 9636172 bytes, checksum: e3e19aa656d9b283ae72bc197a24557a (MD5) Previous issue date: 1998-03-27主0-参1工学_応化分子_分子システム工

Substrate specificity of archaeon Sulfolobus tokodaii biotin protein ligase

Biotin protein ligase (BPL) is an enzyme mediating biotinylation of a specific lysineresidue of the carboxyl carrier protein (BCCP) of biotin-dependent enzymes. We recentlyfound that the substrate specificity of BPL from archaeon Sulfolobus tokodaii is totallydifferent from that of many other organisms, in reflection of a difference in the local sequenceof BCCP surrounding the canonical lysine residue. There is a conserved glycine residue inthe biotin-binding site of Escherichia coli BPL, but this residue is replaced with alanine in S.tokodaii BPL. To test the notion that this substitution dictates the substrate specificity ofthe latter enzyme, this residue, Ala-43, was converted to glycine. The Km values of theresulting mutant, A43G, for substrates, were smaller than those of the wild type, suggestingthat the residue in position 43 of BPL plays an important role in substrate binding

A unique biotin carboxyl carrier protein in archaeon Sulfolobus tokodaii

Biotin carboxyl carrier protein (BCCP) is one subunit or domain of biotin-dependentenzymes. BCCP becomes an active substrate for carboxylation and carboxyl transfer, afterbiotinylation of its canonical lysine residue by biotin protein ligase (BPL). BCCP carries acharacteristic local sequence surrounding the canonical lysine residue, typically -M-K-M-.Archaeon Sulfolobus tokodaii is unique in that its BCCP has serine replaced for themethionine C-terminal to the lysine. This BCCP is biotinylated by its own BPL, but not byEscherichia coli BPL. Likewise, E. coli BCCP is not biotinylated by S. tokodaii BPL,indicating that the substrate specificity is different between the two organisms