We have previously proposed that specific proteins may form insoluble aggregates as a response to an illness-specific proteostatic dysbalance in a subset of brains from individuals with mental illness, as is the case for other chronic brain conditions. So far, established risk factors DISC1 and dysbindin were seen to specifically aggregate in a subset of such patients, as was a novel schizophrenia-related protein, CRMP1, identified through a condition-specific epitope discovery approach. In this process, antibodies are raised against the pooled insoluble protein fractions (aggregomes) of post mortem brain samples from schizophrenia patients, followed by epitope identification and confirmation using additional techniques. Pursuing this epitope discovery paradigm further, we reveal TRIO binding protein (TRIOBP) to be a major substrate of a monoclonal antibody with a high specificity to brain aggregomes from patients with chronic mental illness. TRIOBP is a gene previously associated with deafness which encodes for several distinct protein species, each involved in actin cytoskeletal dynamics. The 3' splice variant TRIOBP-1 is found to be the antibody substrate and has a high aggregation propensity when over-expressed in neuroblastoma cells, while the major 5' splice variant, TRIOBP-4, does not. Endogenous TRIOBP-1 can also spontaneously aggregate, doing so to a greater extent in cell cultures which are post-mitotic, consistent with aggregated TRIOBP-1 being able to accumulate in the differentiated neurons of the brain. Finally, upon expression in Neuroscreen-1 cells, aggregated TRIOBP-1 affects cell morphology, indicating that TRIOBP-1 aggregates may directly affect cell development, as opposed to simply being a by-product of other processes involved in major mental illness. While further experiments in clinical samples are required to clarify their relevance to chronic mental illness in the general population, TRIOBP-1 aggregates are thus implicated for the first time as a biological element of the neuropathology of a subset of chronic mental illness.

As Escherichia coli (E. coli) is well defined with respect to its genome and metabolism, it is a favored host organism for recombinant protein production. However, many processes for recombinant protein production run under suboptimal conditions caused by wrong or incomplete information from an improper screening procedure, because appropriate on‐line monitoring systems are still lacking. In this study, the oxygen transfer rate (OTR), determined on‐line in shake flasks by applying a respiration activity monitoring system (RAMOS) device, was used to characterize the metabolic state of the recombinant organisms. Sixteen clones of E. coli SCS1 with foreign gene sequences, encoding for different target proteins, were cultivated in an autoinduction medium, containing glucose, lactose, and glycerol, to identify relationships between respiration activity and target protein production. All 16 clones showed a remarkably different respiration activity, biomass, and protein formation under induced conditions. However, the clones could be classified into three distinct types, and correlations could be made between OTR patterns and target protein production. For two of the three types, a decrease of the target protein was observed, after the optimal harvest time had passed. The acquired knowledge was used to modify the autoinduction medium to increase the product yield. Additional 1.5 g/L glucose accelerated the production process for one clone, shifting the time point of the maximal product yield from 24 to 17 h. For another clone, lactose addition led to higher volumetric product yields, in fact 25 and 38% more recombinant protein for 2 and 6 g/L additional lactose, respectively. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2012

Allergy is an immunological disorder of the upper airways, lung, skin, and the gut with a growing prevalence over the last decades in Western countries. Atopy, the genetic predisposition for allergy, is strongly dependent on familial inheritance and environmental factors. These observations call for predictive markers of progression from atopy to allergy, a prerequisite to any active intervention in neonates and children (prophylactic interventions/primary prevention) or in adults (immunomodulatory interventions/secondary prevention). In an attempt to identify early biomarkers of the "atopic march" using minimally invasive sampling, CD4+ T cells from 20 adult volunteers (10 healthy and 10 with respiratory allergies) were isolated and quantitatively analyzed and their proteomes were compared in and out of pollen season (+/- antigen exposure). The proteome study based on high-resolution 2D gel electrophoresis revealed three candidate protein markers that distinguish the CD4+ T cell proteomes of normal from allergic individuals when sampled out of pollen season, namely Talin 1, Nipsnap homologue 3A, and Glutamate-cysteine ligase regulatory protein. Three proteins were found differentially expressed between the CD4-1- T cell proteomes of normal and allergic subjects when sampled during pollen season: carbonyl reductase, glutathione S-transferase omega 1, and 2,4-clienoyl-CoA reductase. The results were partly validated by Western blotting.

Pharmaunternehmen wollen schnell spezifische Wirkstoffe entwickeln. Etliche Methoden dafür versprachen mehr als sie halten konnten.

We describe a novel procedure for determination and validation of off-target activities of anti-cluster designation antigen identity 44 (CD44) variant 6 recombinant antibodies by combining two complementary technology platforms; namely UNIchip AV-400, containing a printed serial dilution of CD44 variant 6 and approximately 400 different human proteins, and TISSOMICS, enabling human tissue microarray analysis in high-throughput mode. We have analyzed the performance of two human monoclonal recombinant antibodies directed against CD44 variant 6 protein, BMS 116 and BMS 125, in a blinded study. The antibodies exhibit a clear differentiation with regard to their binding profiles in the two systems. BMS 116 shows a low degree of specificity in the normal human Food and Drug Administration (FDA) tissue panel, which was confirmed by binding to more than 206 proteins on the protein biochip. In contrast, BMS 125 gives a highly selective membranous staining on selected human epithelial tissue components with no off-target activities observed on the protein biochip. Additionally, antibody BMS 125 shows a higher sensitivity to its antigen CD44 variant 6 than antibody BMS 116 as determined by the protein biochip.

AbstractStefan MüllnerUnternehmensgründungen in der Biotechnologie brauchen verständige Gründer, kenntnisreiche Investoren und unterstützende Persönlichkeiten.

This chapter contains sections titled: Introduction Materials and Methods Cells and Culture Conditions In Vitro Activity Testing Affinity Chromatography Electrophoresis and Protein Identification BIAcore Analysis Synthesis of Acyl Cyanides Methyl 5-cyano-5-oxopentanoate Methyl 6-cyano-6-oxohexanoate Methyl-5-cyano-3-methyl-5-oxopentanoate Results Discussion

The human genome has been sequenced and the challenges of understanding the function of the newly discovered genes have been addressed. High-throughput technologies such as DNA microarrays have been developed for the profiling of gene expression patterns in whole organisms or tissues. Protein arrays are emerging to follow DNA chips as possible screening tools. Here, we review the generation and application of microarray technology to obtain more information on the regulation of proteins, their biochemical functions and their potential interaction partners. Already, a large variety of assays based on antibody-antigen interactions exists. In addition, the medical relevance of protein arrays will be discussed.

Not much more than 15 years ago a handful of visionary scientists around the world suggested to sequence and analyze not only the human genome but also as many genomes as possible in order to compare DNA as well as to deduce protein sequences. By that means they expected to get an idea about the organization of life. However, after now having now sequenced the human genome and at least identified around 40,000 genes as coding regions, we are still left with the fundamental questions of how genes are regulated, and what is the rationale of genetic regulatory networks. The basic knowledge and methodologies to elucidate functional regulatory networks of cells and organisms on the protein level had been around for much longer than DNA-based discovery tools. This was mainly due to the fact that proteins have to fulfill universal functions in nature and, unlike DNA polynucleotides, proteins differ not only in their amino acid sequences; they come in nearly all shapes and sizes and have all kinds of physical as well as chemical properties. They can be highly water soluble, e.g., serum and milk proteins, or nearly insoluble in any solvent, e.g., keratin and some other structural proteins. In addition, structure, function, as well as the respective stability of proteins inside and outside of a biological system, are individual features of any given polypeptide. On one hand, the individuality of proteins allows adaptation of any life form to the environment, and on the other it is still a real challenge for biotech R&D and production. The present review is actually the first approach to evaluate and judge the achievements made by Applied Proteome Analysis and Proteomics over the last 27 years.