Soluble Alpha-APP (sAPPalpha) Regulates CDK5 Expression and Activity in Neurons

A growing body of evidence suggests a role for soluble alpha-amyloid precursor protein (sAPPalpha) in pathomechanisms of Alzheimer disease (AD). This cleavage product of APP was identified to have neurotrophic properties. However, it remained enigmatic what proteins, targeted by sAPPalpha, might be involved in such neuroprotective actions. Here, we used high-resolution two- dimensional polyacrylamide gel electrophoresis to analyze proteome changes downstream of sAPPalpha in neurons. We present evidence that sAPPalpha regulates expression and activity of CDK5, a kinase that plays an important role in AD pathology. We also identified the cytoprotective chaperone ORP150 to be induced by sAPPalpha as part of this protective response. Finally, we present functional evidence that the sAPPalpha receptor SORLA is essential to mediate such molecular functions of sAPPalpha in neurons

Automation of phage display fro high-throughput antibody development

Linking a protein displayed on the phage surface (phenotype) to its encoding DNA (genotype, which is integrated into the phage genome) allows us to survey large recombinant Antibodies are important tools for the detection of diagnostic markers and are the most well-known examples of specific molecular interactions. We have developed automated technology that enables the selection of antibodies and other interacting molecules from large recombinant libraries. The physical link between phenotype and genotype in phage display allows selective isolation and amplification of a particular phage encoding a desired antibody fragment. Successive rounds of phage selection, amplification and screening are performed at high throughput, using a pin-based magnetic particle processor. The integration of this with existing DNA and protein array technology enables industrial screening of complex libraries and opens up a new level of functional genomic analysis

High-throughput isolation of recombinant antibodies against recombinant allergens

High-throughput isolation of recombinant !=antibodies against recombinant allergen

Generation and characterization of a Leishmania tarentolae strain for site-directed in vivo biotinylation of recombinant proteins

Leishmania tarentolae is a non-human-pathogenic Leishmania species of growing interest in biotechnology, as it is well-suited for the expression of human recombinant proteins. For many applications it is desirable to express recombinant proteins with a tag allowing easy purification and detection. Hence, we adopted a scheme to express recombinant proteins with a His-tag and, additionally, to site-specifically in vivo biotinylate them for detection. Biotinylation is a relatively rare modification of endogenous proteins that allows easy detection with negligible cross-reactivity. Here, we established a genetically engineered L. tarentolae strain constitutively expressing the codon-optimized biotin-protein ligase from Escherichia coli (BirA). We thoroughly analyzed the strain for functionality using 2-D polyacrylamide-gel electrophoresis (PAGE), mass spectrometry, and transmission electron microscopy (TEM). We could demonstrate that neither metabolic changes (growth rate) nor structural abnormalities (TEM) occurred. To our knowledge, we show the first 2-D PAGE analyses of L. tarentolae. Our results demonstrate the great benefit of the established L. tarentolae in vivo biotinylation strain for production of dual-tagged recombinant proteins. Additionally, 2-D PAGE and TEM results give insights into the biology of L. tarentolae, helping to better understand Leishmania species. Finally, we envisage that the system is transferable to human-pathogenic species

Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays

We describe a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries. The strategy is based on a combination of phage display and high-density arrays. To demonstrate the utility of the method cDNAs of Aspergillus fumigatus cloned into phagemid pJuFo were expressed on the tip of filamentous M13 phage and affinity-selected on solid phase-immobilized serum IgE from allergic patients. Enriched phagemid libraries were amplified in bacteria, plated and arrayed into 384-well microtitre plates by robotic colony picking. cDNA inserts were amplified by high-throughput PCR and gridded onto high-density filter membranes. Filters were iteratively probed with randomly-sequenced inserts until all clones were identified. Eighty-one different sequences encoding IgE-binding proteins likely to cover a large part of the allergen repertoire of the mould were found. This approach represents a widely applicable method for rapid high-throughput identification of all individual cDNAs present in selectively enriched libraries

Array technology and proteomics in autoimmune diseases

Two new technologies (tissue microarrays (TMAs) and proteomics) have generated a great amount of data in life science. High-density TMAs allow for the simultaneous analysis of proteins and RNA by various methods (immunohistochemistry, in situ hybridization, FISH) on a large scale and under highly standardized conditions. Proteomics includes a variety of techniques that are partly high throughput. These techniques aim at the innovation of proteins, the description of the domain structure, the determination of protein sequences and epitope characterization, and ultimately the definition of protein function and protein reactivities in immunologic processes. Proteins that have been characterized accordingly require validation mostly at the morphologic level of defined tissue, linking proteomics to TMAs. In autoimmune diseases, array-based antigenic fingerprinting of autoantibodies will drive the development and the selection of antigen-specific diagnostic tools and therapies. The powerful combination of genomics and proteomics formed in tissue arrays has the potential to change the way the biology of autoimmunity is studied. Novel targets of drug discovery, based on antigen-specific therapies to induce anergy, or regulatory T-cells using the targeted autoantigens of individual patients could be developed in the coming decades