 High Throughput Molecular Biology
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High Throughput Cloning and Expression Methods
The capability to express proteins in heterologous systems is an essential requirement for structural and functional studies of proteins. We are addressing challenging expression problems by application of high throughput technologies to optimize the expression of "insoluble" cytoplasmic, periplasmic proteins and soluble domains of membrane proteins. Our in vivo expression strategy uses E. coli as a host and employs molecular tools containing elements that enable localization to appropriate cellular or extracellular compartments coupled with regulatory elements to permit control and coordination of protein expression. As part of this process, we are also developing high throughput processes for domain-based cloning and expression of high molecular weight proteins and putative soluble domains of membrane proteins. Our strategy is designed to identify the maximum number of targets suitable for bacterial expression before resorting to more time consuming and cost intensive systems.
Development of Genome Scale Expression Strategies > read more
Protein diversity suggests multiple expression strategies will be required to insure production of the highest possible proportion of cellular proteins. We are developing novel cellular and cell-free technologies to optimize the expression of cytoplasmic, periplasmic/secreted proteins and protein domains. These molecular tools contain elements that enable localization to appropriate cellular or extracellular compartments coupled with regulatory elements to permit control and coordination of protein expression. They also incorporate specific fusion components that promote protein stability and solubility or that facilitate detection, purification and/or protein characterization. We describe two approaches for challenging expression targets. One approach describes a high throughput system for the cloning and expression of proteins with signal sequences while the second approach describes domain cloning methods for simple architecture membrane proteins. Both of these approaches have been implemented in cytoplasmic and periplasmic expression systems.
Midwest Center for Structural Genomics - Gene Cloning and Protein Expression > read more
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The Gene Cloning and Protein Expression Core will be responsible for the generation of well-characterized protein expression systems for structural analysis. This core performs two important tasks: (1) production and characterization of expression clones, (2) developing expression constructs and strategies for high-value targets. This core designs PCR primers for gene cloning, selects vectors and strains for cloning and expression, tests protein expression and solubility. To streamline the process, targets for cloning will be grouped by genomes. High-value targets will be exchanged between sites to apply site-specific approaches. The core distributes clones to the Purification and Crystallization Core |
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