This describes the latest modifications to important and original research initially published in the leading methods journal BioTechniques. This new volume brings together, in one collection, some of the best articles published in the exciting field of gene cloning and protein expression and presents updated protocols selected from some of the most widely used techniques practiced in the modern molecular biology laboratory. Specific chapters include the latest information on constructing cDNA libraries, PCR conditions, choosing the correct expression system, and purification strategies.
Table of Contents
Section I: Cloning Vectors and Strategies
1. PCR-Assisted cDNA Cloning: A Guided Tour of the Minefield
2. Positive Selection Vectors to Generate Fused Genes for the Expression of His-Tagged Proteins
3. General Method for Plasmid Construction Using Homologous Recombination
4. Generation of Full-Length cDNA Library from Single Human Prostate Cancer Cells
5. High-Throughput Transformation and Plating Using Petristrips
6. Directional Cloning of Blunt-Ended PCR Products
Section II: Protein Expression and Purification
7. High Copy Number Plasmids Compatible with Commonly Used Cloning Vectors
8. Construct for High-Level Expression and Low Misincorporation of Lysine for Arginine .....
9. UGA Read-Through Artifacts¿When Popular Gene Expression Systems Need a pATCH
10. S. pombe Expression Vector with 6X(His) Tag for Protein Purification and Potential for Ligation-Independent Cloning
11. Purification of Proteins Fused to Either the Amino or Carboxy Terminus of the Mycobacterium xenopi Gyrase A Intein
12. In Vivo Labeling of Over-Expressed Recombinant Proteins in E. coli
13. Recovery of Soluble, Active Recombinant Protein from Inclusion Bodies
14. Simultaneous Expression of Multi-Subunit Proteins in Mammalian Cells .......
15. Isolation of Recombinant Secretory Proteins by Limited Induction and Quantitative Harvest
16. Recovery of Polypeptides Cleaved from Purified Calmodulin-Binding Peptide Fusion Proteins
Section III: Gene Tagging and Epitope Tagging Strategies
17. Mini-Exon Epitope Tagging for Analysis of the Protein Coding Potential of Genomic Sequence
18. Multiple Epitope Tagging of Expressed Proteins for Enhanced Detection
19. Versatile Epitope Tagging Vector for Gene Expression in Mammalian Cells
20. Epitope Tag-Antibody Combination Useful for the Detection of Protein Expression in Prokaryotic and Eukaryotic Cells
21. Use of Staphylococcus aureus Protein-A Subdomains as a Tag for the Sensitive Detection of Recombinant Fusion Proteins
22. Monitoring and Purification of Proteins Using Bovine Papillomavirus E2 Epitope Tags
Section IV: Reporter Gene Technologies
23. Dominant Positive and Negative Selection Using Luciferase, Green Fluorescent Protein and b-Galactosidase Reporter Gene Fusions
24. Quantification of Gene Expression with a Secreted Alkaline Phosphatase Reporter System
25. Application of the Green Fluorescent Protein as a Reporter for Ace1-Based, Two-Hybrid Studies
26. Use of GFP as a Reporter for the Facile Analysis of Sequence-Specific Proteases
27. Applications of Green Fluorescent Protein in Plants
28. Green Fluorescent Protein as a Reporter of Gene Expression and Protein Localization
Section V: Special Purpose Vectors
29. GCN4-Based Expression System (pGES): ........
30. Eukaryotic Conditional Expression System
31. Lac/Tet Dual-Inducible System Functions in Mammalian Cell Lines
32. Cloning Trap for Signal Peptide Sequences
33. Vectors to Target Protein Domains to Different Cellular Compartments
34. Green Fluorescent Protein Labeling of Cytoskeletal Structures ............
35. Establishment of Stable Cell Lines Expressing ........
36. Highly Efficient Eukaryotic Gene Expression Vectors.....
37. Bicistronic Vector for the Creation of Stable Mammalian..........
38. Novel Cloning Method for Recombinant Adenoviruses .........
39. The Admid System: Generation of Recombinant.....
40. Efficient Gene Transfer to Human Endothelial .......
Section VI: In Vitro Protein Expression Technologies
41. Cell-free synthesis and .........
42. E. coli Based In Vitro Transcription......
43. Construction of a Vector .............