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The kinetics of protein folding and binding are not only scientifically relevant to understanding the complex molecular machine-like functionality of proteins inside of cells but can also help elucidate disease pathways and lead to better therapeutic agents. Using nanopores to investigate these kinetics holds great potential for such proteomic studies in which the structure and function of proteins...
Computational structure prediction, including de novo and homology modeling, is an important tool for membrane protein studies. Developing an accurate scoring function that can be used for structure discrimination and assessment remains a challenge. In our previous work, we have analyzed a set of high-resolution membrane protein structures using the network approach developed in our lab and proposed...
Glycoprotein K encoded by the DEV-UL53 gene is one of the DEV envelope glycoproteins. Like most members of the alphaherpesvirinae, the genes in the UL region of its genome are well conserved, which means that the major DEV envelope glycoprotein K possessing similar functions of envelope glycoprotein K of alphaherpesvirinae plays an essential role in viral replication and cell fusion. Glycoprotein...
Non-mitogenic form of recombinant Human acidic fibroblast growth factor (nrhaFGF) retained the cardio and neuro-protective characters of the wild-type aFGF while decreased the potential side effects like mitogenesis activity. In this paper, nrhaFGF was successfully expressed in Escherichia coli and yielded about 5% of the total soluble protein in the 10L fermentation tank. the succeedent chromatography,...
The dispersion of extra-polysaccharide and protein of sludge in sodium pyrophosphate solution (pH=7.0) was studied under the conditions of low mixing speed. The results showed that as the mixing speed were set at 150 and 350 rpm/min, the dispersion of polysaccharide and protein in sodium pyrophosphate solution was mainly controlled by chemical process, and the dispersion efficiency of protein was...
The glycoprotein I (gI) gene homologue of duck plague virus (DPV) was cloned by degenerate polymerase chain reaction (PCR) and sequenced. DPV gI gene open reading frame (ORF) was 1116 bp in length and its primary translation product was a polypeptide of 371 amino acids long. Comparison with other herpesvirus revealed higher similarity, had a close evolution relationship with members of the genus Mardivirus...
Abstract-An open reading frame (ORF) containing 675 nucleotides was got by sequencing the DEV gene libraries constructed by our laboratory. The ORF was identified as DEV UL45 gene by aligning with gene bank database using the software of BlastN and ORF Finder. Specific primers were designed and PCR products containing this ORF were cloned into the vector of PMD18-T. Dot blot confirmed the UL45 gene...
The potentialities of the atomic force microscopy (AFM) make it a tool of the undeniable value for the study of biomolecule samples. One of the main steps for the application of atomic force microscopy to the study of proteins has been the self-assembled monolayers (SAMs). The methods of physical adsorption and chemical modification are utilized to immobilize proteins to substrates efficiently. SAMs...
Our objective is to develop a highly miniaturized, solid-phase platform for assaying the function and inhibition of integral membrane proteins (IMPs). To functionalize materials with active membrane proteins, the challenge is to build stabilized, supported biomembranes in which the substrate to biomembrane spacing can be controlled to accommodate larger membrane protein. The silica microsphere (5...
Our aim is to design alpha-helical peptide complexes to enhance their stability and biological feasibility for the study of membrane proteins and their interactions. In on-going work, we employ (K3A4L2A7L2A3K3) as anchoring molecules, where conjugation of the peptide with fluoresceine isothiocyanate (FITC) allows one to access a variety of chemistries (such as introducing fluorescent dye, etc.) for...
A micromachined polymer membrane-based active probe has been developed for biomolecular force spectroscopy. The probe has integrated but significantly decoupled electrostatic actuation and optical interferometric force sensing capabilities. Devices have been fabricated on silicon substrates using Parylene as the membrane material. The electrostatic actuator integrated into the probe could provide...
Here we present a new technique to monitor dynamic conformational changes in voltage-sensitive membrane-bound proteins using radio frequency (RF) impedance measurements. Xenopus oocytes were transfected to express ShakerB-IR K+ ion channels, and step changes in membrane potential were applied using two-electrode voltage clamp (TEVC). Simultaneously, bipolar extracellular electrodes were used to measure...
A group of membrane proteins endowed with a single COOH-terminal hydrophobic domain capable of insertion into lipid bilayer is known as tail-anchored (TA) proteins. To clarify the insertion mechanism of the TA-domain of human cytochrome b5 (Hcytb5) having various COOH-terminal extensions with bovine opsin sequence, we constructed expression vectors containing membrane-bound form of Hcytb5 (or Hcytb5op(a),...
This paper presents the evaluation of ultra-minimal spout amount from micro-nano pipettes into phospholipid-coated micro-droplets. The pipettes can be used to control the local environment around/inside single cells. Conventionally, the microinjection with pipette has been conducted by air pressures. In this method, the spout of high viscosity solutions is difficult because of the frictional forces...
Actin filaments play central roles in shape determination, cytokinesis, and cell motility. Recently, we found a new type of actin filament that is seemed to contact tightly membrane surface and be buried into membrane under electron microscope. However, the function of this actin filament is not obvious. Then, we have reproduced of such actin filaments using a lipid layer instead of the cell membrane...
Transmembrane proteins are important in biological functions. Proteoliposomes, which contain reconstituted membrane proteins, have been considered to be useful for their investigation. Especially, cell-sized giant liposomes, or giant unilamellar vesicles, are often used for such purposes. Previously, we established a novel method of proteoliposome preparation using membrane fusion between recombinant...
Stearoyl-ACP desaturase (SAD), which catalyzes the first desaturation reaction of the most common pathway of unsaturated fatty acid synthesis in plants, plays a key role in determining the ratio of saturated to unsaturated fatty acids. Using rapid amplification of cDNA ends (RACE) and a peanut cDNA library we had previously constructed, we isolated a 1499 bp cDNA of the SAD gene containing a 1218...
We demonstrate an imaging method combined a terahertz time-domain spectroscopy and an interference effect for label-free protein detection on a membrane filter. Biotin is linked to the membrane using poly ethylene glycol (PEG) or poly ethylene glycol methyl ether (MPEG) to prevent it from being washed off. Binding of the biotin with streptavidin is then observed by measuring the terahertz signal change...
Current cell detection techniques are antibody staining of specific protein markers, morphometric parameters and transgenic markers. These assays are often qualitative and do not quantitatively define the outcome of a cell progression during differentiation. Consequently, we propose to characterize the mechanical behavior of embryonic stem cell, which will predict its stage of differentiation during...
Micro-mixing of highly viscous bio-samples is considered. Existing methods mix bio-samples by only shaking micro-wells or by specially designed micro-stirrers which are ineffective or unapplicable for highly viscous materials, letting alone small volumes at microliter or nanoliter level. Our method mixes viscous bio-samples in micro-wells using orbital shaking plus an innovative block which is built...
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