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Site-specific Modification (site-specific + modification)

Distribution by Scientific Domains
Chemistry66%

Selected Abstracts

Site-Specific Modification of Epstein,Barr Virus-Encoded RNA 1 with N2 -Benzylguanosine Limits the Binding Sites Occupied by PKR

CHEMBIOCHEM, Issue 3 2004
Sujiet Puthenveetil
Examining viral decoys: Epstein,Barr virus (EBV) generates small RNA inhibitors of the human RNA-dependent protein kinase (PKR). We demonstrate that chemical synthesis of analogues of the EBV PKR inhibitor EBER1 bearing single N2 -benzylguanosine substitutions (BnG6 or BnG29) can be used to control the way PKR binds this RNA. [source]

Hybrid linear dendritic macromolecules: From synthesis to applications

JOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 16 2008
Ivan Gitsov
Abstract Linear-dendritic copolymers are intriguing macromolecules, which offer challenge and fascination as purely synthetic objects at the crossroad of organic and polymer chemistry and as promising materials for diverse advanced applications. This review traces their discovery and highlights the synthetic strategies used for their construction. The ambivalent character of the linear-dendritic architecture opens numerous avenues towards emerging and potential applications. Specific solution properties enable the construction of nanometer-sized nanoreactors for reactions in environmentally friendly media, and the creation of "nanosponges" for selective passive binding of fluorescent pH-indicators for environmental or biomonitoring. Another structure,property relationship is used for noncovalent and site-specific modification of glycoproteins, which leads to the formation of "semiartificial" enzymes with enhanced and broadened catalytic activity. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 5295,5314, 2008 [source]

Toward the semisynthesis of multidomain transmembrane receptors: Modification of Eph tyrosine kinases

PROTEIN SCIENCE, Issue 10 2008
Nikhil Singla
Abstract Expressed protein ligation (EPL) is a protein engineering approach that allows the modification or assembly of a target protein from multiple recombinant and synthetic polypeptides. EPL has been previously used to modify intracellular proteins and small integral membrane proteins for structural and functional studies. Here we describe the semisynthetic site-specific modification of the complete, multidomain extracellular regions of both A and B classes of Eph receptor tyrosine kinases. We show that the ectodomains of these receptors can be ligated to different peptides under carefully established experimental conditions, while their biological activity is retained. This work extends the boundaries of the EPL technique for semisynthesis of multidomain, extracellular, disulfide-bonded, and glycosylated proteins and highlights its potential application for reconstituting entire single-pass transmembrane proteins. [source]

Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli

PROTEIN SCIENCE, Issue 9 2004
Jennifer White
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; GPI, glycophosphatidyl inositol; PpDAF, human DAF1,4 expressed in Pichia pastoris, N glycosylated and with an oligohistidine tag; EcDAF, nonglycosylated human DAF 1,4 expressed in Escherichia coli; nDAF, human native glycosylated (GPI-anchored) DAF from erythrocytes; EcDAF-MP, soluble E. coli human DAF linked through a C-terminal cysteine to the myristoylated peptide APT542; PCR, polymerase chain reaction; SCR, short consensus repeat; TCEP, Tris- (2-carboxyethyl) phosphine Abstract Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli -derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential. [source]

Glutathione- S -transferase pi as a model protein for the characterisation of chemically reactive metabolites

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2008
Rosalind E. Jenkins Dr.
Abstract Chemically reactive metabolites (CRMs) are thought to be responsible for a number of adverse drug reactions through modification of critical proteins. Methods that defined the chemistry of protein modification at an early stage would provide invaluable tools for drug safety assessment. Here, human GST pi (GSTP) was exploited as a model target protein to determine the chemical, biochemical and functional consequences of exposure to the hepatotoxic CRM of paracetamol (APAP), N -acetyl- p -benzoquinoneimine (NAPQI). Site-specific, dose-dependent modification of Cys47 in native and His-tagged GSTP was revealed by MS, and correlated with inhibition of glutathione (GSH) conjugating activity. In addition, the adaptation of iTRAQ labelling technology to define precisely the quantitative relationship between covalent modification and protein function is described. Multiple reaction monitoring (MRM)-MS of GSTP allowed high sensitivity detection of modified peptides at physiological levels of exposure. Finally, a bioengineered mutant cytochrome P450 with a broad spectrum of substrate specificities was used in an in vitro reaction system to bioactivate APAP: in this model, GSTP trapped the CRM and exhibited both reduced enzyme activity and site-specific modification of the protein. These studies provide the foundation for the development of novel test systems to predict the toxicological potential of CRMs produced by new therapeutic agents. [source]

Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vector

BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010
Shuohao Huang
Abstract Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase-mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild-type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase-defective retroviral vectors (IDRV), which contains an ATG-deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418-resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site-specific recombination. The successful substitution of the original viral integration machinery with a non-viral mechanism could expand the application of retroviral vectors. Biotechnol. Bioeng. 2010;107:717,729. © 2010 Wiley Periodicals, Inc. [source]