Despite exciting advances in genome sequencing, isolating a protein from its expression system in its native form still presents a complex challenge. In High Throughput Protein Expression and Purification: Methods and Protocols, leading scientists detail the most successful protocols currently in use, including various high throughput cloning schemes, protein expression analysis, and production protocols. This volume describes the use of E. coli, insect, and mammalian cells, as well as cell-free systems for the production of a wide variety of proteins, including glycoproteins and membrane proteins, in order to best represent strategies that create and exploit common features to enable simplified cloning, stable expression, and purification of proteins. Written in the highly successful Methods in Molecular Biology(TM) series format, the chapters present brief introductions to the subject, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and a Notes section for tips on troubleshooting and avoiding known pitfalls.
Cutting-edge and comprehensive, High Throughput Protein Expression and Purification: Methods and Protocols is an ideal reference for protein biochemists and all those who wish to apply these easy-to-use protocols to the many applicable fields.
Part I: Reviews 1. High Throughput Protein Production (HTPP): A Review of Enabling Technologies to Expedite Protein Production Jim Koehn and Ian Hunt 2. Designing Experiments for High-Throughput Protein Expression Stephen P. Chambers and Susanne E. Swalley Part II: Protocols for High-throughput Cloning 3. Gateway Cloning for Protein Expression Dominic Esposito, Leslie A. Garvey, and Chacko S. Chakiath 4. Flexi Vector Cloning Paul G. Blommel, Peter A. Martin, Kory D. Seder, Russell L. Wrobel, and Brian G. Fox 5. The Precise Engineering of Expression Vectors Using High Throughput In-Fusion(TM) PCR Cloning Nick S. Berrow, David Alderton, and Raymond J Owens 6. The Polymerase Incomplete Primer Extension (PIPE) Method Applied to High-Throughput Cloning and Site-Directed Mutagenesis Heath E. Klock and Scott A. Lesley 7. A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins William H. Eschenfeldt, Lucy Stols, Cynthia Sanville Millard, Andrzej Joachimiak, and Mark I. Donnelly 8. System 48 High Throughput Cloning and Protein Expression Analysis James M. Abdullah, Andrzej Joachimiak, and Frank R. Collart Part III: Protocols for High-throughput Protein Expression and Purification 9. Automated 96-Well Purification of Hexahistidine-Tagged Recombinant Proteins on MagneHis Ni2+-Particles Chiann-Tso Lin, Priscilla A. Moore, and Vladimir Kery 10. E.coli and Insect Cell Expression, Automated Purification and Quantitative Analysis Stephen P. Chambers, John R. Fulghum, Douglas A. Austen, Fan Lu, and Susanne E. Swalley 11. Hexahistidine-Tagged Maltose-Binding Protein as a FusionPartner for the Production of Soluble Recombinant Proteins in Escherichia coli Brian P. Austin, Sreedevi Nallamsetty, and David S. Waugh 12. PHB-Intein-Mediated Protein Purification Strategy Alison R. Gillies, Mahmoud Reza Banki, and David W. Wood 13. High-Throughput Biotinylation of Proteins Brian K. Kay, Sang Thai, and Veronica V. Volgina 14. High-Throughput Insect Cell Protein Expression Applications Mirjam Buchs, Ernie Kim, Yann Pouliquen, Michael Sachs, Sabine Geisse, Marion Mahnke, and Ian Hunt 15. High-Throughput Protein Expression Using Cell-free System Kalavathy Sitaraman and Deb K. Chatterjee 16. The Production of Glycoproteins by Transient Expression in Mammalian Cells Joanne E. Nettleship, Nahid Rahman-Huq, and Raymond J. Owens 17. High-Throughput Expression and Detergent Screening of Integral Membrane Proteins Said Eshaghi 18. Cell-Free Expression for Nanolipoprotein Particles (NLPs): Building a High-Throughput Membrane Protein Solubility Platform Jenny A. Cappuccio, Angela K. Hinz, Edward A. Kuhn, Julia Fletcher, Erin S. Arroyo, Paul T. Henderson, Craig D. Blanchette, Vicki L. Walsowrth, Michele H. Corzett, Richard J. Law, Joseph B. Pesavento, Brent W. Segelke, Todd A. Sulchek, Brett A. Chromy, Federico Katzen, Todd Peterson, Graham Bench, Wieslaw Kudlicki, Paul D. Hoeprich Jr., and Matthew A. Coleman 19. Expression and Purification of Soluble His6-Tagged TEV Protease Joseph E. Tropea, Scott Cherry, and David S. Waugh 20. High-Throughput Protein Concentration and Buffer Exchange: Comparison of Ultrafiltration and Ammonium Sulfate Precipitation Priscilla A. Moore and Vladimir Kery