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Variable Domain (variable + domain)
Selected AbstractsRecombinant anti-hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and core-,(1,3)-fucose residuesPLANT BIOTECHNOLOGY JOURNAL, Issue 4 2004Rajan Sriraman Summary Plant-based expression systems are attractive for the large-scale production of pharmaceutical proteins. However, glycoproteins require particular attention as inherent differences in the N-glycosylation pathways of plants and mammals result in the production of glycoproteins bearing core-xylose and core-,(1,3)-fucose glyco-epitopes. For treatments requiring large quantities of repeatedly administered glycoproteins, the immunological properties of these non-mammalian glycans are a concern. Recombinant glycoproteins could be retained within the endoplasmic reticulum (ER) to prevent such glycan modifications occurring in the late Golgi compartment. Therefore, we analysed cPIPP, a mouse/human chimeric IgG1 antibody binding to the ,-subunit of human chorionic gonadotropin (hCG), fused to a C-terminal KDEL sequence, to investigate the efficiency of ER retrieval and the consequences in terms of N-glycosylation. The KDEL-tagged cPIPP antibody was expressed in transgenic tobacco plants or Agrobacterium -infiltrated tobacco and winter cherry leaves. N-Glycan analysis showed that the resulting plantibodies contained only high-mannose (Man)-type Man-6 to Man-9 oligosaccharides. In contrast, the cPIPP antibody lacking the KDEL sequence was found to carry complex N-glycans containing core-xylose and core-,(1,3)-fucose, thereby demonstrating the secretion competence of the antibody. Furthermore, fusion of KDEL to the diabody derivative of PIPP, which contains an N-glycosylation site within the heavy chain variable domain, also resulted in a molecule lacking complex glycans. The complete absence of xylose and fucose residues clearly shows that the KDEL-mediated ER retrieval of cPIPP or its diabody derivative is efficient in preventing the formation of non-mammalian complex oligosaccharides. [source] Factors contributing to decreased protein stability when aspartic acid residues are in ,-sheet regionsPROTEIN SCIENCE, Issue 7 2002P.R. Pokkuluri Abstract Asp residues are significantly under represented in ,-sheet regions of proteins, especially in the middle of ,-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a ,-domain. Two Gln residues of the immunoglobulin light-chain variable domain (VL) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a ,-strand, and that of Q89D, located in the middle of a ,-strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type VL -Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, VL -Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type VL -Len domain. The structures of mutants VL -Len Q38D and VL -Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 Å resolution. We found no major perturbances in the structures of these Q,D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a ,-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a ,-strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo. [source] Change in dimerization mode by removal of a single unsatisfied polar residue located at the interfacePROTEIN SCIENCE, Issue 9 2000P. R. Pokkuluri Abstract The importance of unsatisfied hydrogen bonding potential on protein-protein interaction was studied. Two alternate modes of dimerization (conventional and flipped form) of an immunoglobulin light chain variable domain (VL) were previously identified. In the flipped form, interface residue Gln89 would have an unsatisfied hydrogen bonding potential. Removal of this Gln should render the flipped dimer as the more favorable quaternary form. High resolution crystallographic studies of the Q89A and Q89L mutants show, as we predicted, that these proteins indeed form flipped dimers with very similar interfaces. A small cavity is present in the Q89A mutant that is reflected in the ,100 times lower association constant than found for the Q89L mutant. The association constant of Q89A and Q89L proteins (4 × 106 M,1 and > 108 M,1) are 10- and 1,000-fold higher than that of the wild-type protein that forms conventional dimers clearly showing the energetic reasons for the flipped dimer formation. [source] scFv-based fluorogen activating proteins and variable domain inhibitors as fluorescent biosensor platformsBIOTECHNOLOGY JOURNAL, Issue 9 2009Crystal N. Falco Abstract Single chain antibodies (scFvs) are engineered proteins composed of IgG variable heavy (VH) and variable light (VL) domains tethered together by a flexible peptide linker. We have characterized the individual VH or VL domain activities of several scFvs isolated from a yeast surface-display library for their ability to bind environmentally sensitive fluorogenic dyes causing them to fluoresce. For many of the scFvs, both VH and VL domains are required for dye binding and fluorescence. The analysis of other scFvs, however, revealed that either the VH or the VL domain alone is sufficient to cause the fluorogenic dye activation. Furthermore, the inactive complementary domains in the original scFvs either contribute nothing to, or actually inhibit the activity of these active single domains. We have explored the interactions between active variable domains and inactive complementary domains by extensive variable domain swapping through in vitro gene manipulations to create hybrid scFvs. In this study, we demonstrate that significant alteration of the fluorogenic dye activation by the active VH or VL domains can occur by partnering with different VH or VL complementary domains in the scFv format. Hybrid scFvs can be generated that have fluorogen-activating domains that are completely inhibited by interactions with other domains. Such hybrid scFvs are excellent platforms for the development of several types of genetically encoded, fluorescence-generating biosensors. [source] Method for generation of human hyperdiversified antibody fragment libraryBIOTECHNOLOGY JOURNAL, Issue 1 2007Philippe Mondon Abstract The selection of antibody fragments from libraries using in vitro screening technologies has proven to be a very good alternative to the classical hybridoma technology, and has overcome the laborious process of antibody humanization. However, the complexity of the library is critical in the probability of being able to directly isolate a high affinity antibody specific to a target. We report a method to make hyperdiversified antibody fragment libraries, based on human immunoglobulin variable genes mimicking the somatic hypermutation process. This mutagenesis technology, MutaGenÔ, was used for the first time on the entire variable domain (frameworks and CDRs) of large repertoires of human variable antibody domains. Our MutaGenÔ process uses low-fidelity human polymerases, known as mutases, suggested to be involved in the somatic hypermutation process of immunoglobulin genes. Depending on the mutases used, we generated complementary mutation patterns with randomly distributed mutations. The libraries were generated with an average of 1.8 mutations per 100 amino acids. The hyperdiversified antibody fragment libraries constructed with our process should enable the selection of antibody fragments specific to virtually any target. [source] Freshwater selenium-methylating bacterial thiopurine methyltransferases: diversity and molecular phylogenyENVIRONMENTAL MICROBIOLOGY, Issue 2 2005S. Favre-Bonté Summary The diversity of bacterial thiopurine methyltransferases (bTPMT) among five natural Se-methylating freshwaters was investigated by polymerase chain reaction (PCR) screenings and sequencings. DNA sequence analyses confirmed the cloned products' identity and revealed a broad diversity of freshwater TPMTs. Neighbour-joining (NJ) phylogenetic analyses combining these sequences, all GenBank entries closely related to these sequences and deduced TPMTs obtained in this work from selected ,-proteobacteria showed TPMTs to form a distinct radiation, closely related to UbiG methyltransferases. Inside the TPMT phylogenetic cluster, eukaryote sequences diverged early from the bacterial ones, and all the bacterial database entries belonged to a subgroup of ,-proteobacteria, with an apparent lateral transfer of a particular allele to ,-proteobacteria of Bordetella. The NJ phylogenetic tree revealed 22 bTPMT lineages, 10 of which harboured freshwater sequences. All lineages showed deep and long branches indicative of major genetic drifts outside regions encoding highly conserved domains. Selected residues among these highly variable domains could reflect adaptations for particular ecological niches. PCR lineage-specific primers differentiated Se-methylating freshwaters according to their ,tpm lineage' signatures. Most freshwater tpm alleles were found to be distinct from those available in the databases, but a group of tpm was found encoding TPMTs identical to an Aeromonas veronii TPMT characterized in this work. [source] Existence of front solutions for a nonlocal transport problem describing gas ionizationMATHEMATICAL METHODS IN THE APPLIED SCIENCES, Issue 12 2010M. Günther Abstract We discuss a moving boundary problem arising from a model of gas ionization in the case of negligible electron diffusion and suitable initial data. It describes the time evolution of an ionization front. Mathematically, it can be considered as a system of transport equations with different characteristics for positive and negative charge densities. We show that only advancing fronts are possible and prove short-time well posedness of the problem in Hölder spaces of functions. Technically, the proof is based on a fixed-point argument for a Volterra-type system of integral equations involving potential operators. It crucially relies on estimates of such operators with respect to variable domains in weighted Hölder spaces and related calculus estimates. Copyright © 2010 John Wiley & Sons, Ltd. [source] scFv-based fluorogen activating proteins and variable domain inhibitors as fluorescent biosensor platformsBIOTECHNOLOGY JOURNAL, Issue 9 2009Crystal N. Falco Abstract Single chain antibodies (scFvs) are engineered proteins composed of IgG variable heavy (VH) and variable light (VL) domains tethered together by a flexible peptide linker. We have characterized the individual VH or VL domain activities of several scFvs isolated from a yeast surface-display library for their ability to bind environmentally sensitive fluorogenic dyes causing them to fluoresce. For many of the scFvs, both VH and VL domains are required for dye binding and fluorescence. The analysis of other scFvs, however, revealed that either the VH or the VL domain alone is sufficient to cause the fluorogenic dye activation. Furthermore, the inactive complementary domains in the original scFvs either contribute nothing to, or actually inhibit the activity of these active single domains. We have explored the interactions between active variable domains and inactive complementary domains by extensive variable domain swapping through in vitro gene manipulations to create hybrid scFvs. In this study, we demonstrate that significant alteration of the fluorogenic dye activation by the active VH or VL domains can occur by partnering with different VH or VL complementary domains in the scFv format. Hybrid scFvs can be generated that have fluorogen-activating domains that are completely inhibited by interactions with other domains. Such hybrid scFvs are excellent platforms for the development of several types of genetically encoded, fluorescence-generating biosensors. [source] N-linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgGBIOTECHNOLOGY PROGRESS, Issue 1 2009Patrick H. C. van Berkel Abstract We studied the variations in N-linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one of the six different antibodies. Antibody expression was based on glutamine synthetase selection technology in suspension growing CHO-K1SV cells. The glycans detected on the Fc fragment were mainly of the core-fucosylated complex type containing zero or one galactose and little to no sialic acid. The glycosylation was highly consistent for the same cell line when grown multiple times, indicating the robustness of the production and glycan analysis procedure. However, a twofold to threefold difference was observed in the level of galactosylation and/or non-core-fucosylation between the 105 different cell lines, suggesting clone-to-clone variation. These differences may change the Fc-mediated effector functions by such antibodies. Large variation was also observed in the oligomannose-5 glycan content, which, when present, may lead to undesired rapid clearance of the antibody in vivo. Statistically significant differences were noticed between the various glycan parameters for the six different antibodies, indicating that the variable domains and/or light chain isotype influence Fc glycosylation. The glycosylation altered when batch production in shaker was changed to fed-batch production in bioreactor, but was consistent again when the process was scaled from 400 to 5,000 L. Taken together, the observed clone-to-clone glycosylation variation but batch-to-batch consistency provides a rationale for selection of optimal production cell lines for large-scale manufacturing of biopharmaceutical human IgG. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||