Gene cloning and utility phophorylation assay of a protein-fused substrate for a highly sensitive detection of cdc2 protein kinase using a radioisotope detection technique for the development of a protein biochip

Ko, Kyong-Cheol,Choi, Mi Hee,Park, Sang Hyun John Wiley Sons, Ltd. 2009 Journal of labelled compounds & radiopharmaceutica Vol.52 No.4

<P>The prototype of the cdc2 protein kinase in mammalian cells regulates its entry into mitosis by phosphorylating a group of key proteins in the major cell cycle transitions. In this study, using the mep45 gene encoding the 45 kDa major envelope protein (Mep45) of Selenomonas ruminantium, a rumen bacteria, a Mep45-fused substrate (PKTPKKAKKL-Mep45, MFS-cdc2) was cloned to detect the activity of cdc2 protein kinase. We report here on a strategy for the detection of a phosphorylation of a substrate catalyzed by cdc2 protein kinase by using a radioisotope detection technique. It is possible to constantly obtain a reasonable quantity of MFS-cdc2 for the cdc2 protein kinase assay and its cost can be as low as a synthesized peptide. Results of the study indicate that the Mep45-fused protein can be used effectively as a substrate for detecting the activity of cdc2 protein kinase and it can be used in developing a protein biochip for a high-throughput screening and also for studying protein–protein interactions. Copyright © 2009 John Wiley & Sons, Ltd.</P> <B>Graphic Abstract</B> <P>Gene cloning and utility phophorylation assay of a protein-fused substrate for a highly sensitive detection of cdc2 protein kinase using a radioisotope detection technique for the development of a protein biochip Kyong-Cheol Ko, Mi Hee Choi, Sang Hyun Park<SUP>*</SUP>Using the mep45 gene encoding the 45 kDa major envelope protein (Mep45) of Selenomonas ruminantium, a rumen bacteria, a Mep45-fused substrate (PKTPKKAKKL-Mep45, MFS-cdc2) was cloned to detect the activity of cdc2 protein kinase. We report here on a strategy for the detection of a phosphorylation of a substrate catalyzed by cdc2 protein kinase by using a radioisotope detection technique. Copyright © 2009 John Wiley & Sons, Ltd. <img src='wiley_img/03624803-2009-52-4-JLCR1579-gra001.gif' alt='wiley_img/03624803-2009-52-4-JLCR1579-gra001'> </P>

Multiple detection of proteins by SERS-based immunoassay with core shell magnetic gold nanoparticles

Shin, M.H.,Hong, W.,Sa, Y.,Chen, L.,Jung, Y.J.,Wang, X.,Zhao, B.,Jung, Y.M. Elsevier 2014 Vibrational spectroscopy Vol.72 No.-

A highly selective and sensitive surface-enhanced Raman scattering (SERS)-based immunoassay for the multiple detection of proteins has been developed. The proposed core shell magnetic gold (Au) nanoparticles allow for successful protein separation and high SERS enhancement for protein detection. To selectively detect a specific protein in a mixed protein solution, we employed the sandwich type SERS immunoassay with core shell magnetic Au nanoparticles utilizing specific antigen-antibody interactions. Based on this proposed SERS immunoassay, we can successfully detect proteins in very low concentrations (~800ag/mL of mouse IgG and ~5fg/mL of human IgG) with high reproducibility. Magnetically assisted protein separation and detection by this proposed SERS immunoassay would provide great potential for effective and sensitive multiple protein detection. This technique allows for the straightforward SERS-based bioassays for quantitative protein detections.

Protein Patterns on a Vaginal Mucus during Spontaneous and Estrus Synchronization using CIDR in Korean Native Cattle (Hanwoo)

정학재,김남국,윤현일,이석동,권혁진,고진성,오해령,최성복,최연호,이휘철 사단법인 한국동물생명공학회 2008 한국동물생명공학회지 Vol.23 No.4

The aim of the present recent study was to compare the protein patterns in the vaginal mucus of Hanwoo cattles during spontaneous and CIDR induced-estrus. Ten cattles, who had been observed in estrus, received no treatment and served as the group of cattles with normal spontaneous estrus. Thirteen cattles in the CIDR received an CIDR insert on day14 were removed and cattles were injected GnRH on day 15. Vaginal mucus samples were collected from all cattles at the same time the single AI in cattles with spontaneous estrus and the AI in cattles with induced estrus. Spontaneous and CIDR-induced estrus vaginal mucus samples were analyzed on two different array surfaces: cation-exchange (CM10), anion-exchange (Q10). In addition, using the NaCl solution by which the proteins combined after washing are 0.5, 1 and 2 M, it was fractionated and a protein was collected successively. The results are summarized as follows: 1) Ionic surfaces chemistries (Q10 and CM10) gave the best results in terms of detectable protein peaks, with more than 100 protein peaks in the two fractions and under each condition. 2) Protein mass spectrometer using 11 different proteins in protein identification of 7 were able to determine the protein. List of identified proteins as follows; Ribosome-binding protein 1, GRIP 1-associated protein 1, Katanin p60 ATPase-containing subunit A-like 1, Protein FAM44A, DUF729 domain-containing protein 1, Prolactin precursor, Dihydrofolate erductase. Conclusively, on the basis of this study, protein expression in the vaginal mucus could be used as an indicator for time of estrus manifestation in order to increase conception rates by applying AI at an optional time.

Ligand immobilization on polydiacetylene-coated and surface-enhanced Raman scattering-encoded beads for label-free detection

Kim, Hyung-Mo,Kang, Yoo-Lee,Chung, Woo-Jae,Kyeong, San,Jeong, Sinyoung,Kang, Homan,Jeong, Cheolhwan,Rho, Won-Yeop,Kim, Dong-Hyuk,Jeong, Dae Hong,Lee, Yoon-Sik,Jun, Bong-Hyun Elsevier 2015 Journal of industrial and engineering chemistry Vol.21 No.-

<P><B>Abstract</B></P> <P>A better understanding of protein–protein interactions can be obtained from multiplex protein detection technologies, and spectrally encoded beads can provide fast and efficient means for this type of detection methods. However, high-throughput detection is challenging due to the requirement of using labeled secondary proteins to detect protein binding events. We have previously reported that polydiacetylene-coated surface-enhanced Raman scattering-encoded beads (PDA–SERS beads) can provide an enhanced encoding capacity owing to their SERS properties as well as their potential for label-free detection from the PDA layer. In this study, we introduced ligands to the PDA–SERS beads by using methods for making free-floating vesicles and planar solid substrates, which enabled the detection of target proteins by PDA fluorescence in a PDA–SERS beads system. By using PDA–SERS beads immobilized with biotin, the fluorescence intensities of biotin-conjugated PDA–SERS beads were increased with an increase in the concentration of streptavidin. And, we could detect 2×10<SUP>−8</SUP> M of streptavidin by measuring the fluorescence intensity without the requirement of an additional labeling step.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Generation of Efficient Fingerprint for GFP-like Fold and Computational Identification of Potential GFP-like Homologs

Selvakumar Edwardraja,Ganapathiraman Munussami,Amit Goyal,이선구 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.6

There is a considerable interest in the detection of GFP-like proteins due to their structural stability and functional usefulness. GFP-like proteins share highly conserved beta-barrel fold with 11 beta-strands. However, their low sequence identity hampers efficient identification of their homologous proteins from database. In this study, an attempt was made to generate a fingerprint for efficient detection of GFP-like proteins. Overlapped conserved residues (OCR)-based approach has been used to design a protein fingerprint based on sequentially and structurally conserved residues in secondary structures to detect homologous proteins very efficiently. Therefore, a fingerprint for GFP-like fold was designed using the OCR approach. However, its specificity was too low to be used for the identification of novel proteins. The conserved residues of loop regions were added and optimized to improve its specificity without losing its high sensitivity. The optimized fingerprint was employed to scan NR database. A total of 20 hypothetical proteins were detected, among which nine were validated as potential GFP-like homologs.

Fabrication of Disposable Protein Chip for Simultaneous Sample Detection

Lee, Chang-Soo,Lee, Sang-Ho,Kim, Yun-Gon,Oh, Min-Kyu,Hwang, Taek-Sung,Rhee, Young-Woo,Song, Hwan-Moon,Kim, Bo-Yeol,Kim, Yong-Kweon,Kim, Byung-Gee The Korean Society for Biotechnology and Bioengine 2006 Biotechnology and Bioprocess Engineering Vol.11 No.5

In this study, we have described a method for the fabrication of a protein chip on silicon substrate using hydrophobic thin film and microfluidic channels, for the simultaneous detection of multiple targets in samples. The use of hydrophobic thin film provides for a physical, chemical, and biological barrier for protein patterning. The microfluidic channels create four protein patterned strips on the silicon surfaces with a high signal-to-noise ratio. The feasibility of the protein chips was determined in order to discriminate between each protein interaction in a mixture sample that included biotin, ovalbumin, hepatitis B antigen, and hepatitis C antigen. In the fabrication of the multiplexed assay system, the utilization of the hydrophobic thin film and the microfluidic networks constitutes a more convenient method for the development of biosensors or biochips. This technique may be applicable to the simultaneous evaluation of multiple protein-protein interactions.

Application of Engineered Zinc Finger Proteins Immobilized on Paramagnetic Beads for Multiplexed Detection of Pathogenic DNA

( Jiyoung Shim ),( Langley Williams ),( Dohyun Kim ),( Kisung Ko ),( Moon-soo Kim ) 한국미생물 · 생명공학회 2021 Journal of microbiology and biotechnology Vol.31 No.9

Micro-scale magnetic beads are widely used for isolation of proteins, DNA, and cells, leading to the development of in vitro diagnostics. Efficient isolation of target biomolecules is one of the keys to developing a simple and rapid point-of-care diagnostic. A zinc finger protein (ZFP) is a doublestranded (ds) DNA-binding domain, providing a useful scaffold for direct reading of the sequence information. Here, we utilized two engineered ZFPs (Stx2-268 and SEB-435) to detect the Shiga toxin (stx2) gene and the staphylococcal enterotoxin B (seb) gene present in foodborne pathogens, Escherichia coli O157 and Staphylococcus aureus, respectively. Engineered ZFPs are immobilized on a paramagnetic bead as a detection platform to efficiently isolate the target dsDNA-ZFP bound complex. The small paramagnetic beads provide a high surface area to volume ratio, allowing more ZFPs to be immobilized on the beads, which leads to increased target DNA detection. The fluorescence signal was measured upon ZFP binding to fluorophore-labeled target dsDNA. In this study, our system provided a detection limit of ≤ 60 fmol and demonstrated high specificity with multiplexing capability, suggesting a potential for development into a simple and reliable diagnostic for detecting multiple pathogens without target amplification.

Ligand immobilization on polydiacetylene-coated and surfaceenhanced Raman scattering-encoded beads for label-free detection

김형모,강유리,정우재,경산,정신영,강호만,정철환,노원엽,김동혁,정대홍,이윤식,전봉현 한국공업화학회 2015 Journal of Industrial and Engineering Chemistry Vol.21 No.1

A better understanding of protein–protein interactions can be obtained from multiplex protein detectiontechnologies, and spectrally encoded beads can provide fast and efficient means for this type of detectionmethods. However, high-throughput detection is challenging due to the requirement of using labeledsecondary proteins to detect protein binding events.We have previously reported that polydiacetylenecoatedsurface-enhanced Raman scattering-encoded beads (PDA–SERS beads) can provide an enhancedencoding capacity owing to their SERS properties as well as their potential for label-free detection fromthe PDA layer. In this study, we introduced ligands to the PDA–SERS beads by using methods for makingfree-floating vesicles and planar solid substrates, which enabled the detection of target proteins by PDAfluorescence in a PDA–SERS beads system. By using PDA–SERS beads immobilized with biotin, thefluorescence intensities of biotin-conjugated PDA–SERS beads were increased with an increase in theconcentration of streptavidin. And, we could detect 2 10 8M of streptavidin by measuring thefluorescence intensity without the requirement of an additional labeling step.

Detection and discrimination of <i>Shigella sonnei</i> and <i>Shigella flexneri</i> based on vacuolar responses in <i>Saccharomyces cerevisiae</i>

Nguyen, Ngoc-Tu,Park, Ra-Mi,Kim, Yang-Hoon,Min, Jiho Elsevier 2018 Journal of biotechnology Vol.287 No.-

<P><B>Abstract</B></P> <P>This study provided a system for bacteria detection based on a lysosome-like-vacuole response in the yeast <I>Saccharomyces cerevisiae</I>. Vacuoles are factors known to activate the immune system in the presence of foreign substances. Here, <I>Shigella sonnei</I> and <I>Shigella flexneri</I> were exposed to yeast to analyze the alteration of vacuolar enzymes. The ability to detect the bacteria was evaluated by confocal microscopy after exposing and staining vacuoles with LysoTracker. Results showed that the treatment of yeast with these bacteria increased the number of red vacuole-like organelles surrounding yeast nuclei. Thus, vacuole alteration can be used as a biomarker for bacteria detection. Next, the expression of vacuolar enzymes under the influence of bacteria was examined using two-dimensional gel electrophoresis (2-DE) method for screening specific biomarkers for each <I>Shigella</I> strain. Finally, the recombinant yeasts that contained biomarkers fused to different fluorescent proteins confirmed the ability of yeast to detect these two <I>Shigella</I> strains at concentrations ranging from 10 to 100 CFU/mL.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Detection of <I>Shigella</I> strains was performed using vacuolar response in <I>S. cerevisiae</I>. </LI> <LI> The vacuolar proteomic in response to <I>Shigella</I> was analyzed for screening of specific biomarkers. </LI> <LI> Biomarker for each strain was fused with different fluorescent proteins for the distinct detection. </LI> <LI> The ability of recombinant yeasts to recognize <I>Shigella</I> strains individually from 10 CFU/ml. </LI> </UL> </P>

His-tagged protein immobilization on cationic ferrite magnetic nanoparticles

박성진,김승연,김승훈,박경민,황병희 한국화학공학회 2018 Korean Journal of Chemical Engineering Vol.35 No.6

Magnetic nanoparticles have been applied in various fields because of their interesting magnetic properties. Immobilization on magnetic nanoparticles is a very important step in functionalizing them. We examined protein immobilization efficiency using interactions between his-tagged enhanced green fluorescence protein and affordable cationic ferrite magnetic nanoparticles for the first time. Four types of ferrite magnetic nanoparticles were verified: cobalt iron oxide, copper iron oxide, nickel iron oxide, and iron (III) oxide as negative controls. Among the four ferrite magnetic nanoparticles, copper ferrite magnetic nanoparticle was confirmed to have the highest immobilization efficiency at 3.0mg proteins per gram ferrite magnetic nanoparticle and 78% of total enhanced green fluorescence protein. In addition, the maximum binding efficiency was determined for copper ferrite magnetic nanoparticle. Consequently, this newly verified his-tag-immobilizing capacity of copper ferrite magnetic nanoparticle could provide a facile, capable, and promising strategy for immobilizing his-tagged proteins or peptides with high purity for biosensors, magnetic separation, or diagnostics.