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High-throughput System (high-throughput + system)
Selected AbstractsHigh-throughput system for determining dissolution kinetics of inclusion bodiesBIOTECHNOLOGY JOURNAL, Issue 5 2009Astrid Dürauer Dr. Abstract Efficient solubilization is a crucial step during inclusion body processing and dissolving conditions were usually empirically established. Here we describe a new methodology for rapid screening of solubilization conditions and evaluation of dissolution kinetics in microtiter plates. Increase of protein in solution over time was directly related to decrease of turbidity measured by absorbance at 600 nm. Dissolution kinetics of inclusion bodies were described by a first-order reaction kinetics, which was used for drug dissolution modeling. Reaction constants were in the range of 0.01,0.03 s,1 for buffer conditions providing sufficient solubilization power. This method is not limited to the screening of optimal buffer conditions for solubilization and can be applied for studying other parameters involved in the solubility of IBs, such as pI of the protein, influence of fermentation conditions, influence of initial protein concentration, and more. [source] Update of the molecular basis of familial hypercholesterolemia in The Netherlands,HUMAN MUTATION, Issue 6 2005Sigrid W. Fouchier Abstract Autosomal-dominant hypercholesterolemia (ADH) has been identified as a major risk factor for coronary vascular disease (CVD) and is associated with mutations in the low-density lipoprotein receptor (LDLR) and the apolipoprotein B (APOB) gene. Since 1991 DNA samples from clinically diagnosed ADH patients have been routinely analyzed for the presence of LDLR and APOB gene mutations. As of 2001, 1,641 index patients (164 index patients per year) had been identified, while from 2001 onward a more sensitive, high-throughput system was used, resulting in the identification of 1,177 new index patients (average=294 index patients per year). Of these 1,177 index cases, 131 different causative genetic variants in the LDLR gene and six different causative mutations in the APOB gene were new for the Dutch population. Of these 131 mutations, 83 LDLR and four APOB gene mutations had not been reported before. The inclusion of all 2,818 index cases into the national screening program for familial hypercholesterolemia (FH) resulted in the identification of 7,079 relatives who carried a mutation that causes ADH. Screening of the LDLR and APOB genes in clinically diagnosed FH patients resulted in approximately 77% of the patients being identified as carriers of a causative mutation. The population of patients with ADH was divided into three genetically distinct groups: carriers of an LDLR mutation (FH), carriers of an APOB mutation (FDB), and non- LDLR/non- APOB patients (FH3). No differences were found with regard to untreated cholesterol levels, response to therapy, and onset of CVD. However, all groups were at an increased risk for CVD. Therefore, to ultimately identify all individuals with ADH, the identification of new genes and mutations in the genes that cause ADH is of crucial importance for the ongoing national program to identify patients with ADH by genetic cascade screening. Hum Mutat 26(6), 550,556, 2005. © 2005 Wiley-Liss, Inc. [source] A novel mammalian display system for the selection of protein,protein interactions by decoy receptor engagementJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2004Peter Ellmark Abstract The emerging field of proteomics has created a need for new high-throughput methodologies for the analysis of gene products. An attractive approach is to develop systems that allow for clonal selection of interacting protein pairs from large molecular libraries. In this study, we have characterized a novel approach for identification and selection of protein,protein interactions, denoted SPIRE (selection of protein interactions by receptor engagement), which is based on a mammalian expression system. We have demonstrated proof of concept by creating a general plasma membrane bound decoy receptor, by displaying a protein or a peptide genetically fused to a trunctated version of the CD40 molecule. When this decoy receptor is engaged by a ligand to the displayed protein/peptide, the receptor expressing cell is rescued from apoptosis. To design a high-throughput system with a highly parallel capacity, we utilized the B cell line WEHI-231, as carrier of the decoy receptor. One specific peptide-displaying cell could be identified and amplified, based on a specific receptor engagement, in a background of 12,500 wild-type cells after four selections. This demonstrates that the approach may serve as a tool in post-genomic research for identifying protein,protein interactions, without prior knowledge of either component. Copyright © 2004 John Wiley & Sons, Ltd. [source] Twenty-four-well plate miniature bioreactor high-throughput system: Assessment for microbial cultivationsBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2007Kevin Isett Abstract High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (µ)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the µ-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6,±,2.4, 46.5,±,4.6, 51.6,±,3.7, and 56.1,±,1.6 h,1 at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively. Biotechnol. Bioeng. 2007;98: 1017,1028. © 2007 Wiley Periodicals, Inc. [source] Identification of a ligand for IgG-Fc derived from a soluble peptide library based on fusion proteins secreted by S. cerevisiaeBIOTECHNOLOGY JOURNAL, Issue 6 2007Christa Mersich Abstract Biological libraries are important tools in the development of new peptide-based compounds. Here, we describe the use of a soluble peptide library system as a complementary tool in the field of ligand development. Random peptides were expressed in S. cerevisiae as carboxy-terminal extensions of the eukaryotic initiation factor 5a (eIF5a) and secreted into the culture supernatant. Expression and screening of this library were performed in a microwell format. As an example of this versatile approach, we describe the identification of a ligand for the human IgG-Fc fragment. Ligands binding IgG-Fc show great potential in a wide variety of applications including development of therapeutics, streamlining the large-scale purification of antibodies, and applications in diagnostic tests. We demonstrated the utility of this system. After screening only 6160 clones, we identified a ligand with the peptide sequence of TRRRTCSPPTWPRARARSTPSGCSSTGPSANRG. An affinity constant of 3.9 x 105 M -1 was determined by a biosensor method. Handling and maintenance of this library is conceptually simple and highly applicable for automated high-throughput systems. [source] | ||||||||||