García-Fruitós Insoluble Proteins

Methods and Protocols
2015
ISBN: 978-1-4939-2205-5
Verlag: Springer US
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band 1258, 425 Seiten, eBook

Reihe: Methods in Molecular Biology

With insolubility proving to be one of the most crippling bottlenecks in the protein production and purification process, this volume serves to aid researchers working in the recombinant protein production field by describing a wide number of protocols and examples. Insoluble Proteins: Methods and Protocols includes chapters that describe not only the recombinant protein production in different expression systems but also different purification and characterization methods to finally obtain these difficult-to-obtain proteins. Beginning with protein production methods using both prokaryotic and eukaryotic expression systems, the book continues with purification protocols using insoluble proteins, the characterization of insoluble proteins, as well as a general overview of interesting applications of insoluble proteins. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and practical, Insoluble Proteins: Methods and Protocols aims to provide the scientific community with detailed and reliable state-of-the-art protocols that are used in order to successfully produce and purify recombinant proteins prone to aggregate.

Zielgruppe

Professional/practitioner

Autoren/Hrsg.

García-Fruitós, Elena

Inhaltsverzeichnis

General Introduction: Recombinant Protein Production and Purification of Insoluble Proteins.- Overcoming the Solubility Problem in E. coli : Available Approaches for Recombinant Protein Production.- Optimization of Culture Parameters and Novel Strategies to Improve Protein Solubility.- Cleavable Self-Aggregating Tags (cSAT) for Protein Expression and Purification.- Beyond the Cytoplasm of Escherichia coli : Localizing Recombinant Proteins Where You Want Them.- Characterization of Amyloid-Like Properties in Bacterial Intracellular Aggregates.- Co-Translational Stabilization of Insoluble Proteins in Cell-Free Expression Systems.- Functional Expression of Plant Membrane Proteins in Lactococcus lactis. - High Cell-Density Expression System: Yeast Cells in a Phalanx Efficiently Produce a Certain Range of “Difficult-to-Express” Secretory Recombinant Proteins.- Insect Cells-Baculovirus System for the Production of Difficult to Express Proteins.- Transient Expression in HEK 293 Cells: An Alternative to E. coli for the Production of Secreted and Intracellular Mammalian Proteins.- Recombinant Glycoprotein Production in Human Cell Lines.- Soluble Recombinant Protein Production in Pseudoaltermonas haloplanktis TAC125.- A Screening Methodology for Purifying Proteins with Aggregation Problems.- Solubilization and Refolding of Inclusion Body Proteins.- Bacterial Inclusion Body Purification.- Characterization of Intracellular Aggresomes by Fluorescent Microscopy.- Dialysis: A Characterization Method of Aggregation Tendency.- Applications of Mass Spectrometry to the Study of Protein Aggregation.- Insoluble Protein Assemblies Characterized by Fourier Transform Infrared Spectroscopy.- Insoluble Protein Characterization by Circular Dichroism (CD) Spectroscopy and Nuclear Magnetic Resonance (NMR).- Methods for Characterization of Protein Aggregates.- Predicting the Solubility of Recombinant Proteins in Escherichia coli .- Insoluble ProteinApplications: The Use of Bacterial Inclusion Bodies as Biocatalysts.