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Full Description
This volume serves to aid researchers working in the recombinant protein production field by describing a wide number of protocols and examples. Chapters describe recombinant protein production in different expression systems, prokaryotic and eukaryotic expression systems, purification protocols, characterization of insoluble proteins and a general overview of interesting applications of insoluble proteins. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and cutting-edge, Insoluble Proteins: Methods and Protocols, Second Edition aims to be a useful practical guide to researches to help further their study in this field.
Contents
General Introduction: Recombinant protein production and purification of insoluble proteins.- Overcoming the solubility problem in E. coli: available approaches for recombinant protein production.- A3D 2.0 update for the prediction and optimization of protein solubility.- Tailoring codon usage to the underlying biology for protein expression optimization.- Protein Expression Optimization Strategies in E. coli: A Tailored Approach in Strain Selection and Parallelizing Expression Conditions.- A novel approach for production of aggregation prone proteins using the spidroin-derived NT* tag.- Cleavable Self-Aggregating Tags (cSAT) for Therapeutic Peptide Expression and Purification.- Use Intein Cleavable Polyhydroxyalkanoate Synthase Fusions to Improve Protein Solubility.- Introduction of a hexalysine (6K) tag can protect from N-terminal cleavage and increase yield of recombinant proteins expressed in the periplasm of E. coli.- Two-tiered selection and screening strategy to increase functional enzyme production in Escherichia coli.- Method for production of cysteine-rich proteins in Lactococcus lactis expression system.- Heterologous protein production in Lactobacillus (plantarum) using pSIP vectors.- Soluble recombinant protein production in Pseudoaltermonas haloplanktis TAC125: the case study of the full-length human CDKL5 protein.- A protocol to enhance soluble protein expression in the cytoplasm of Bacillus subtilis.- Applications of cell-free synthesized membrane protein precipitates.- 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: from expression screening for soluble constructs to protein quality control.- Transient gene expression in human Expi293 cells.- Recombinant Expression of Complex Proteins in Human Cells Lines.- Optimizing chaperone removal strategy from over-expressed recombinant proteins: GNE, a case study.- Intrinsically disordered proteins (IDP): purification under denaturing conditions.- Solubilization and Refolding of Inclusion Body Proteins.- Non-denaturing solubilization of Inclusion Bodies from Lactic Acid Bacteria.- Purification of Inclusion Bodies Produced in Bacteria and Yeast.- Eukaryotic aggresomes: protocols and tips for their production, purification and handling.- Characterization of the conformational properties of soluble and insoluble proteins by Fourier transform infrared spectroscopy.- Characterizing Soluble Protein Aggregates using Native Mass Spectrometry Coupled with Temperature-control Electrospray Ionization and Size-exclusion Chromatography.- Quality control of proteins solubilized from inclusion bodies.- Methods for the Characterizationof Protein Aggregates.- Insoluble Protein Applications: The Use of Bacterial Inclusion Bodies as Biocatalysts.- Methods for Processing Protein Aggregates into Surfaces.
