| 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
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 83 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Bachelor | 21 | 25% |
| Student > Master | 10 | 12% |
| Researcher | 8 | 10% |
| Student > Ph. D. Student | 4 | 5% |
| Student > Doctoral Student | 4 | 5% |
| Other | 6 | 7% |
| Unknown | 30 | 36% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 29 | 35% |
| Agricultural and Biological Sciences | 6 | 7% |
| Immunology and Microbiology | 6 | 7% |
| Pharmacology, Toxicology and Pharmaceutical Science | 3 | 4% |
| Unspecified | 3 | 4% |
| Other | 6 | 7% |
| Unknown | 30 | 36% |