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Tools of the Trade: Developing Antibody-Based Detection Capabilities for Recombinant Proteins

Protein-specific antibodies serve as critical tools for detection, quantification, and characterization of recombinant proteins. Perhaps the most important and widely used antibody-based procedures for recombinant protein applications are Western immunoblotting ...

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A Matter of Packaging: Influence of Nucleosome Positioning on Heterologous Gene Expression

The organization of DNA into the various levels of chromatin compaction is the main obstacle that restricts the access of transcriptional machinery to genes. Genome-wide chromatin analyses have shown that there are common chromatin organization patterns for most genes but have also re ...

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Routine Identity Confirmation of Recombinant Proteins by MALDI-TOF Mass Spectrometry

Peptide mass fingerprinting (PMF) by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides a simple and direct means to unequivocally confirm identity of recombinant proteins based on predicted peptide profiles. Many univ ...

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Using Folding Promoting Agents in Recombinant Protein Production: A Review

Recombinant production has become an invaluable tool for supplying research and therapy with proteins of interest. The target proteins are not in every case soluble and/or correctly folded. That is why different production parameters such as host, cultivation conditions and co-expr ...

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Regulated Recombinant Protein Production in the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125

This review reports results from our laboratory on the development of an effective inducible expression system for the homologous/heterologous protein production in cold-adapted bacteria. Recently, we isolated and characterized a regulative genomic region from Pseudoalt ...

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Heat-Shock Protein Fusion Vectors for Improved Expression of Soluble Recombinant Proteins in Escherichia coli

Molecular chaperones or heat-shock proteins (HSPs) are protein machines that interact with unfolded or partially folded polypeptides and assist them in attaining their proper conformation. The folding reaction relies on a complex array of scaffolding effects and ATP-driven conf ...

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Plasmid DNA Production for Therapeutic Applications

Plasmid DNA (pDNA) is the base for promising DNA vaccines and gene therapies against many infectious, acquired, and genetic diseases, including HIV-AIDS, Ebola, Malaria, and different types of cancer, enteric pathogens, and influenza. Compared to conventional vaccines, DNA vaccines h ...

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Heterologous High-Level Gene Expression in the Photosynthetic Bacterium Rhodobacter capsulatus

The functional expression of heterologous genes in standard hosts such as Escherichia coli is often hampered by various limitations including improper folding, incomplete targeting, and missassembly of the corresponding enzymes. This observation led to the development of num ...

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A New Bacterial Co-expression System for Over-expressing Soluble Protein and Validating ProteinProtein Interaction

Toxic, membrane, and hydrophobic proteins are usually difficult to individually over-express in Escherichia coli because they require a binding-partner protein for folding and stability. To obtain these types of soluble proteins or protein complexes, protein co-expression is u ...

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A Novel Strategy for the Construction of Genomic Mutants of the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125

The sequencing and the annotation of the marine Antarctic Pseudoalteromonas haloplanktis TAC125 genome has paved the way to investigate on the molecular mechanisms involved in adaptation to cold conditions. The growing interest in this unique bacterium prompted the developing of ...

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Gene Expression Profiling of Tissues and Cell Lines: A Dual-Color Microarray Method

Since its origin in the mid-1990s, gene expression profiling by microarray has become a productive and useful tool in basic science and preclinical research. Current dual-color, high-density cDNA oligo arrays contain 60-mer detectors for the whole human genome. With this powerful techn ...

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Identification of a Common Variant Affecting Human Episodic Memory Performance Using a Pooled Genome-Wide Association Approach: A Case Study of Diseas

Genome-wide association studies (GWAS) are an important tool for discovering novel genes associated with disease or traits. Careful design of case–control groups greatly facilitates the efficacy of these studies. Here we describe a pooled GWAS study undertaken to find novel genes ass ...

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Integrative Systems Biology Approaches to Identify and Prioritize Disease and Drug Candidate Genes

Although a number of computational approaches have been developed to integrate data from multiple sources for the purpose of predicting or prioritizing candidate disease genes, relatively few of them focus on identifying or ranking drug targets. To address this deficit, we have develo ...

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RNAi-Based Functional Pharmacogenomics

Experimental alteration of gene expression is a powerful technique for functional characterization of disease genes. RNA interference (RNAi) is a naturally occurring mechanism of gene regulation, which is triggered by the introduction of double-stranded RNA into a cell. This phen ...

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Genetic Predisposition to -Thalassemia and Sickle Cell Anemia in Turkey: A Molecular Diagnostic Approach

The thalassemia syndromes are a diverse group of inherited disorders that can be characterized according to their insufficient synthesis or absent production of one or more of the globin chains. They are classified in to α, β, γ, δβ, δ, and εγδβ thalassemias depending on the globin chain(s) affected. ...

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Bar-Coded, Multiplexed Sequencing of Targeted DNA Regions Using the Illumina Genome Analyzer

To date, genome-wide association (GWA) studies, in which thousands of markers throughout the genome are simultaneously genotyped, have identified hundreds of loci underlying disease susceptibility. These regions typically span 5–100 kb, and resequencing efforts to identify po ...

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Targeted SNP Genotyping Using the TaqMan Assay

More than 99% of genomic DNA sequence is identical among humans, and not surprisingly, slight variations in sequence can often produce a major effect on phenotype. Sequence variants may also mediate the manner in which humans are susceptible to disease or respond to environmental factors such ...

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Methods for MicroRNA Microarray Profiling

MicroRNAs (miRNAs) are single-stranded, small RNA molecules of 21–23 nucleotides that are primarily involved in the regulation of gene expression. A number of recent studies have shown that miRNAs play a vital role in signaling pathways important for human oncogenesis and may hold promise ...

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Allelic Expression Profiling to Dissect Genome-Wide Association Study Signals

Genome-wide association studies are providing exciting new insight into the genetics of complex disease, but oftentimes, the genomic regions associated with the trait of interest are large enough to contain several equally plausible candidate genes. Commonly, no obvious, putativ ...

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Genomic Analysis by Oligonucleotide Array Comparative Genomic Hybridization Utilizing Formalin-Fixed, Paraffin-Embedded Tissues

Formalin fixation has been used to preserve tissues for more than a hundred years, and there are currently more than 300 million archival samples in the United States alone. The application of genomic protocols such as high-density oligonucleotide array Comparative Genomic Hybridiza ...

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