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Protein Crystallography

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Cover of 'Protein Crystallography'

Table of Contents

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    Book Overview
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    Chapter 1 Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag
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    Chapter 2 Protein Crystallization
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    Chapter 3 Advanced Methods of Protein Crystallization
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    Chapter 4 The “Sticky Patch” Model of Crystallization and Modification of Proteins for Enhanced Crystallizability
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    Chapter 5 Crystallization of Membrane Proteins: An Overview
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    Chapter 6 Locating and Visualizing Crystals for X-Ray Diffraction Experiments
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    Chapter 7 Collection of X-Ray Diffraction Data from Macromolecular Crystals
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    Chapter 8 Identifying and Overcoming Crystal Pathologies: Disorder and Twinning
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    Chapter 9 Applications of X-Ray Micro-Beam for Data Collection
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    Chapter 10 Serial Synchrotron X-Ray Crystallography (SSX)
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    Chapter 11 Time-Resolved Macromolecular Crystallography at Modern X-Ray Sources
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    Chapter 12 Structure Determination Using X-Ray Free-Electron Laser Pulses
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    Chapter 13 Processing of XFEL Data
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    Chapter 14 Many Ways to Derivatize Macromolecules and Their Crystals for Phasing
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    Chapter 15 Experimental Phasing: Substructure Solution and Density Modification as Implemented in SHELX
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    Chapter 16 Contemporary Use of Anomalous Diffraction in Biomolecular Structure Analysis
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    Chapter 17 Long-Wavelength X-Ray Diffraction and Its Applications in Macromolecular Crystallography
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    Chapter 18 Acknowledging Errors: Advanced Molecular Replacement with Phaser
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    Chapter 19 Rosetta Structure Prediction as a Tool for Solving Difficult Molecular Replacement Problems
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    Chapter 20 Radiation Damage in Macromolecular Crystallography
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    Chapter 21 Boxes of Model Building and Visualization
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    Chapter 22 Structure Refinement at Atomic Resolution
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    Chapter 23 Low Resolution Refinement of Atomic Models Against Crystallographic Data
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    Chapter 24 Stereochemistry and Validation of Macromolecular Structures
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    Chapter 25 Validation of Protein–Ligand Crystal Structure Models: Small Molecule and Peptide Ligands
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    Chapter 26 Protein Data Bank (PDB): The Single Global Macromolecular Structure Archive
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    Chapter 27 Databases, Repositories, and Other Data Resources in Structural Biology
Attention for Chapter 1: Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag
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Chapter title
Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag
Chapter number 1
Book title
Protein Crystallography
Published in
Methods in molecular biology, January 2017
DOI 10.1007/978-1-4939-7000-1_1
Pubmed ID
Book ISBNs
978-1-4939-6998-2, 978-1-4939-7000-1
Authors

Sreejith Raran-Kurussi, David S. Waugh

Editors

Alexander Wlodawer, Zbigniew Dauter, Mariusz Jaskolski

Abstract

Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His6- or a dual His6-MBP tagged fusion protein by Gateway(®) recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His6 tag or a His6-MBP tag can be made on the basis of this solubility test.

Mendeley readers

The data shown below were compiled from readership statistics for 20 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 20 100%

Demographic breakdown

Readers by professional status Count As %
Student > Bachelor 6 30%
Student > Master 4 20%
Researcher 3 15%
Student > Ph. D. Student 2 10%
Other 2 10%
Other 2 10%
Unknown 1 5%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 9 45%
Agricultural and Biological Sciences 4 20%
Immunology and Microbiology 2 10%
Pharmacology, Toxicology and Pharmaceutical Science 1 5%
Nursing and Health Professions 1 5%
Other 2 10%
Unknown 1 5%