Role of Nucleic Acid and Protein Manipulation Technologies in High-throughput Structural Biology Efforts

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 Keywords: affinity tag; Arabidopsis thaliana; bioinformatics; catabolite repression; codon bias; codon usage; Escherichia coli; expression vectors; factor Xa; fusion protein; gene expression; heterologous expression; high-density oligonucleotide arrays; Laboratory Information Management Systems; MALDI-TOF; mass spectrometry; peptide mapping; PreScission; protease cleavage; protein sequence analysis; solubility tag; soluble protein; structure and functional biology; TEV; T7 bacteriophage promoter; T7 RNA polymerase; target selection; thrombin; transcriptome