Puchheim, Germany

Sloning BioTechnology

Puchheim, Germany
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Karlstetter M.,University of Regensburg | Walczak Y.,University of Regensburg | Weigelt K.,University of Regensburg | Weigelt K.,Erasmus University Rotterdam | And 5 more authors.
Journal of Immunology | Year: 2010

Microgliosis is a common phenomenon in neurodegenerative disorders, including retinal dystrophies. To identify candidate genes involved in microglial activation, we used DNA-microarray analysis of retinal microglia from wild-type and retinoschisin-deficient (Rs1h-/Y) mice, a prototypic model for inherited retinal degeneration. Thereby, we cloned a novel 76 aa protein encoding a microglia/macrophage-restricted whey acidic protein (WAP) termed activated microglia/macrophage WAP domain protein (AMWAP). The gene consists of three exons and is located on mouse chromosome 11 in proximity to a chemokine gene cluster. mRNA expression of AMWAP was detected in microglia from Rs1h-/Y retinas, brain microglia, and other tissue macrophages. AMWAP transcription was rapidly induced in BV-2 microglia upon stimulation with multiple TLR ligands and IFN-γ. The TLR-dependent expression of AMWAP was dependent on NF-κB, whereas its microglia/macrophage-specific transcription was regulated by PU.1. Functional characterization showed that AMWAP overexpression reduced the proinflammatory cytokines IL-6 and IL-1β and concomitantly increased expression of the alternative activation markers arginase 1 and Cd206. Conversely, small interfering RNA knockdown of AMWAP lead to higher IL-6, IL-1β, and Ccl2 transcript levels, whereas diminishing arginase 1 and Cd206 expression. Moreover, AMWAP expressing cells had less migratory capacity and showed increased adhesion in a trypsin-protection assay indicating antiserine protease activity. In agreement with findings from other WAP proteins, micromolar concentrations of recombinant AMWAP exhibited significant growth inhibitory activity against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. Taken together, we propose that AMWAP is a counter-regulator of proinflammatory microglia/macrophage activation and a potential modulator of innate immunity in neurodegeneration. Copyright © 2010 by The American Association of Immunologists, Inc.

Zhai W.,Pfizer | Glanville J.,Pfizer | Fuhrmann M.,Sloning BioTechnology | Mei L.,Pfizer | And 15 more authors.
Journal of Molecular Biology | Year: 2011

We present a method for synthetic antibody library generation that combines the use of high-throughput immune repertoire analysis and a novel synthetic technology. The library design recapitulates positional amino acid frequencies observed in natural antibody repertoires. V-segment diversity in four heavy (V H) and two kappa (V κ) germlines was introduced based on the analysis of somatically hypermutated donor-derived repertoires. Complementarity-determining region 3 length and amino acid designs were based on aggregate frequencies of all V H and V κ sequences in the data set. The designed libraries were constructed through an adaptation of a novel gene synthesis technology that enables precise positional control of amino acid composition and incorporation frequencies. High-throughput pyrosequencing was used to monitor the fidelity of construction and characterize genetic diversity in the final 3.6 × 10 10 transformants. The library exhibited Fab expression superior to currently reported synthetic approaches of equivalent diversity, with greater than 93% of clones observed to successfully display both a correctly folded heavy chain and a correctly folded light chain. Genetic diversity in the library was high, with 95% of 7.0 × 10 5 clones sequenced observed only once. The obtained library diversity explores a comparable sequence space as the donor-derived natural repertoire and, at the same time, is able to access novel recombined diversity due to lack of segmental linkage. The successful isolation of low- and subnanomolar-affinity antibodies against a diverse panel of receptors, growth factors, enzymes, antigens from infectious reagents, and peptides confirms the functional viability of the design strategy. © 2011 Elsevier Ltd. All rights reserved.

Kuhn S.M.,TU Munich | Rubini M.,TU Munich | Fuhrmann M.,Sloning BioTechnology | Theobald I.,TU Munich | Skerra A.,TU Munich
Journal of Molecular Biology | Year: 2010

We describe a strategy for the rapid selection of mutant aminoacyl-tRNA synthetases (aaRS) with specificity for a novel amino acid based on fluorescence-activated cell sorting of transformed Escherichia coli using as reporter the enhanced green fluorescent protein (eGFP) whose gene carries an amber stop codon (TAG) at a permissive site upstream of the fluorophore. To this end, a one-plasmid expression system was developed encoding an inducible modified Methanocaldococcus jannaschii (Mj) tyrosyl-tRNA synthetase, the orthogonal cognate suppressor tRNA, and eGFPUAG in an individually regulatable fashion. Using this system a previously described aaRS with specificity for O-methyl-L-tyrosine (MeTyr) was engineered for 10-fold improved incorporation of the foreign amino acid by selection from a mutant library, prepared by error-prone as well as focused random mutagenesis, for MeTyr-dependent eGFP fluorescence. Applying alternating cycles of positive and negative fluorescence-activated bacterial cell sorting in the presence or in the absence, respectively, of the foreign amino acid was crucial to select for high specificity of MeTyr incorporation. The optimized synthetase was used for the preparative expression of a modified uvGFP carrying MeTyr at position 66 as part of its fluorophore. This biosynthetic protein showed quantitative incorporation of the non-natural amino acid, as determined by mass spectrometry, and it revealed a unique emission spectrum due to the altered chemical structure of its fluorophore. Our combined genetic/selection system offers advantages over earlier approaches that relied wholly or in part on antibiotic selection schemes, and it should be generally useful for the engineering and optimization of orthogonal aaRS/tRNA pairs to incorporate non-natural amino acids into recombinant proteins. © 2010 Elsevier Ltd.

Sloning BioTechnology | Date: 2011-01-04

Chemicals for use in industry, science, agriculture, horticulture and forestry, namely, carrier materials in the nature of polymer and copolymer plates, spheres, polymer beads, and copolymer beads; enzymes, namely, enzymes for scientific and research purposes; libraries of nucleic acids for laboratory use; bulk material, namely, vaccine adjuvants, and vaccine stabilizers for vaccines for use in the manufacture of pharmaceutical vaccines; biotechnology formed genes and gene sequences, used for gene optimization, protein expression, enzyme and protein engineering, synthetic biology, directed evolution, mutation analysis, genetic engineering. Pharmaceutical, veterinary, and sanitary preparations, namely, enzyme preparations for use in enzyme therapy; nucleic acids for pharmaceutical or veterinary purposes; libraries of nucleic acids for pharmaceutical or veterinary purposes; genes for pharmaceutical or veterinary purposes; gene sequences for pharmaceutical or veterinary purposes; nucleic acid sequences and chemical reagents for medicinal and veterinary purposes; preparations for destroying vermin; fungicides; herbicides; vaccines. Pharmaceutical research and development; agricultural research; scientific and industrial research and development; development of data processing programs by order of third parties; chemist services; biologist, biochemist, and biotechnologist research services; development of new technology for others in the fields of genomics and biotechnology, namely, genetic engineering services, molecular biologist services, gene synthesis services, gene mutation analysis and optimization, protein expression, selection of ribozymes, protein interaction analysis, reading frame control, and diagnosis using nucleic acids, in particular DNA.

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