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1.
Mao H Graziano JJ Chase TM Bentley CA Bazirgan OA Reddy NP Song BD Smider VV 《Nature biotechnology》2010,28(11):1195-1202
Antibody discovery typically uses hybridoma- or display-based selection approaches, which lack the advantages of directly screening spatially addressed compound libraries as in small-molecule discovery. Here we apply the latter strategy to antibody discovery, using a library of ~10,000 human germline antibody Fabs created by de novo DNA synthesis and automated protein expression and purification. In multiplexed screening assays, we obtained specific hits against seven of nine antigens. Using sequence-activity relationships and iterative mutagenesis, we optimized the binding affinities of two hits to the low nanomolar range. The matured Fabs showed full and partial antagonism activities in cell-based assays. Thus, protein drug leads can be discovered using surprisingly small libraries of proteins with known sequences, questioning the requirement for billions of members in an antibody discovery library. This methodology also provides sequence, expression and specificity information at the first step of the discovery process, and could enable novel antibody discovery in functional screens. 相似文献
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Hutchins BM Kazane SA Staflin K Forsyth JS Felding-Habermann B Schultz PG Smider VV 《Journal of molecular biology》2011,406(4):595-303
Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe-Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well-defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe-Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical “handle” by which immunoconjugates and multimers can be engineered. 相似文献
4.
Aruna Kasoju M Lakshmi Narasu Charuvaka Muvva Bathula VV SubbaRao 《Bioinformation》2012,8(14):684-686
Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse
consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a
protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server.
The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the
backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the
3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer
molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of
inhibition of aflatoxin. 相似文献
5.
Failure of Hairpin-Ended and Nicked DNA To Activate DNA-Dependent Protein Kinase: Implications for V(D)J Recombination 总被引:3,自引:1,他引:2
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Vaughn Smider W. Kimryn Rathmell Greg Brown Susanna Lewis Gilbert Chu 《Molecular and cellular biology》1998,18(11):6853-6858
V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PKCS or p460). Ku directs DNA-PKCS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PKCS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PKCS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself. 相似文献
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Kim D Rhee Y Rhodes D Sharma V Sorenson O Greener A Smider V 《Journal of molecular biology》2005,351(4):763-775
Genes can be mutated by altering DNA content (base changes) or DNA length (insertions or deletions). Most in vitro directed evolution processes utilize nucleotide content changes to produce DNA libraries. We tested whether gain of function mutations could be identified using a mutagenic process that produced only nucleotide deletions. Short nucleotide stretches were deleted in a plasmid encoding lacZ, and screened for increased beta-galactosidase activity. Several mutations were found in the origin of replication that quantitatively and qualitatively altered plasmid behavior in vivo. Some mutations allowed co-residence of ColE1 plasmids in Escherichia coli, and implicate hairpin structures II and III of the ColE1 RNA primer as determinants of plasmid compatibility. Thus, useful and unexpected mutations can be found from libraries containing only deletions. 相似文献
7.
Visualization of molecular structures aids in the understanding of structural and functional roles of biological macromolecules.
Macromolecular transport between the cell nucleus and cytoplasm is facilitated by the nuclear pore complex (NPC). The ring
structure of the NPC is large and contains several distinct proteins (nucleoporins) which function as a selective gate for the passage
of certain molecules into and out of the nucleus. In this note we demonstrate the utility of a python code that allows direct
mapping of the physiochemical properties of the constituent nucleoporins on the scaffold of the yeast NPC׳s cytoplasmic view. We
expect this tool to be useful for researchers to visualize the NPC based on their physiochemical properties and how it alters when
specific mutations are introduced in one or more of the nucleoporins. The code developed using Python is available freely from the
authors. 相似文献
8.
Jonathan W. Day Chan Hyuk Kim Vaughn V. Smider Peter G. Schultz 《Bioorganic & medicinal chemistry letters》2013,23(9):2598-2600
The bidentate metal binding amino acid bipyridylalanine (BpyAla) was incorporated into a disulfide linked cyclic peptide phage displayed library to identify metal ion binding peptides. Selection against Ni2+–nitrilotriacetic acid (NTA) enriched for sequences containing histidine and BpyAla. BpyAla predominated when selections were carried out at lower pH, consistent with the differential pKa’s of histidine and BpyAla. Two peptides containing BpyAla were synthesized and found to bind Ni2+ with low micromolar dissociation constants. Incorporation of BpyAla and other metal binding amino acids into peptide and protein libraries should enable the evolution of novel binding and catalytic activities. 相似文献
9.
Trisler K Looger LL Sharma V Baker M Benson DE Trauger S Schultz PG Smider VV 《The Journal of biological chemistry》2007,282(36):26344-26353
Antibody affinity is critically important in therapeutic applications, as well as steady state diagnostic assays. Picomolar affinity antibodies, approaching the association limit of protein-protein interactions, have been discovered for highly potent antigens, but even such high-affinity binders have off-rates sufficient to negate therapeutic efficacy. To cross this affinity threshold, antibodies that tether their targets in a manner other than reversible non-covalent interaction will be required. Here we report the design and construction of an antibody that forms an irreversible complex with a protein antigen in a metal-dependent reaction. The complex resists thermal and chemical denaturation, as well as attempts to remove the coordinating metal ion. Such irreversibly binding antibodies could facilitate the development of next generation "reactive antibody" therapeutics and diagnostics. 相似文献
10.
VV. R. PHILIPSON F.L.S. 《Botanical journal of the Linnean Society. Linnean Society of London》1987,95(1):19-25
Problems presented by genera, or small groups of genera, which have been given family rank are reviewed, and the genera are divided into a number of categories according to the closeness of their affinity to other genera or families. Satellite genera that stand in close relation to families should be united with them. Binary families, that have been divided into two (or more) related families, should be re–united. Families connected by linking genera, should, logically, be united but practical considerations usually prevent this. Clusters of diverse but more or less distantly related genera present unusual problems, being treated either as several, often monogeneric families or as a loosely structured family. Truly isolated genera must be given family and often ordinal rank. 相似文献