The Center for Eukaryotic Structural Genomics (CESG) was founded as a collaborative effort to develop technologies for the rapid and economic determination of protein three-dimensional structures. The initial focus was on the genome of the model plant Arabidopsis thaliana. Protocols for high-throughput cloning of Arabidopsis open reading frames into Escherichia coli expression vectors are presented along with an analysis of results from approximately 2000 cloning experiments. Open reading frames were chosen on the likelihood that they would represent important unknown regions of protein conformation and fold space or that they would elucidate novel fold-function relationships. The chosen open reading frames were amplified from a cDNA pool created by reverse transcription of RNA isolated from an Arabidopsis callus culture. A novel Gateway protocol was developed to insert the amplified open reading frames into an entry vector for storage and sequence determination. Sequence verified entry clones were then used to create expression vectors again via the Gateway system.
Introduction
Overview of Structural Genomics Initiatives World‐wide
Use of Bioinformatics as a Predictive Tool
High‐throughput Cloning Strategies Applied to
Arabidopsis
cDNA Sources
DNA Chip Technologies as a Predictive Tool
Genes Lacking Introns
Restriction Digestion and Ligation
Topoisomerase Cloning
Transformation
Sequence Verification
Escherichia coli
Expression Systems
T7 Promoter Systems
Arabinose Operon Promoter System
Wheat Germ Cell‐free Translation Systems
Characterization of Expression, Solubility, and Purity
Electrophoresis and Dye Binding
ELISA‐based Detection
S‐Tag‐Based Detection
Protein Tags and Multi‐step Purification Procedures
Role of Protease Cleavage in Target Preparation
Protein Characterization by Mass Spectrometry
Integration with Broader Biological Sciences Community
Acknowledgments
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