High Throughput Protein Expression and Purification

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™ 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.

Provides step-by-step protocols for protein production that utilize a wide range of cutting-edge techniques Covers cell-based and cell-free methodologies needed to produce bacterial proteins, eukaryotic proteins, soluble and membrane proteins Examines a variety of cloning methodologies that enable high-throughput cloning for protein production Describes the use of a variety of tags to facilitate parallel processing for high-throughput protein production Includes several reviews to help readers choose which protocols best suits their needs Provides protocols that utilize robotics for sample handling, as well as protocols designed small laboratories performing experiments by hand

Inhalt
High-Throughput Protein Production (HTPP): A Review of Enabling Technologies to Expedite Protein Production.- Designing Experiments for High-Throughput Protein Expression.- Gateway Cloning for Protein Expression.- Flexi Vector Cloning.- The Precise Engineering of Expression Vectors Using High-Throughput In-Fusion™ PCR Cloning.- The Polymerase Incomplete Primer Extension (PIPE) Method Applied to High-Throughput Cloning and Site-Directed Mutagenesis.- A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins.- System 48 High-Throughput Cloning and Protein Expression Analysis.- Automated 96-Well Purification of Hexahistidine-Tagged Recombinant Proteins on MagneHis Ni2+-Particles.- E. coli and Insect Cell Expression, Automated Purification and Quantitative Analysis.- Hexahistidine-Tagged Maltose-Binding Protein as a Fusion Partner for the Production of Soluble Recombinant Proteins in Escherichia coli.- PHB-Intein-Mediated Protein Purification Strategy.- High-Throughput Biotinylation of Proteins.- High-Throughput Insect Cell Protein Expression Applications.- High-Throughput Protein Expression Using Cell-Free System.- The Production of Glycoproteins by Transient Expression in Mammalian Cells.- High-Throughput Expression and Detergent Screening of Integral Membrane Proteins.- Cell-Free Expression for Nanolipoprotein Particles: Building a High-Throughput Membrane Protein Solubility Platform.- Expression and Purification of Soluble His6-Tagged TEV Protease.- High-Throughput Protein Concentration and Buffer Exchange: Comparison of Ultrafiltration and Ammonium Sulfate Precipitation.