Chapter title |
Expression and Purification of Active Recombinant Human Alpha-1 Antitrypsin (AAT) from Escherichia coli
|
---|---|
Chapter number | 19 |
Book title |
Alpha-1 Antitrypsin Deficiency
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-7163-3_19 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7161-9, 978-1-4939-7163-3
|
Authors |
Beena Krishnan, Lizbeth Hedstrom, Daniel N. Hebert, Lila M. Gierasch, Anne Gershenson |
Abstract |
Well-established genetic manipulation procedures along with a fast doubling time, the ability to grow in inexpensive media, and easy scaleup make Escherichia coli (E. coli) a preferred recombinant protein expression platform. Human alpha-1 antitrypsin (AAT) and other serpins are easily expressed in E. coli despite their metastability and complicated topology. Serpins can be produced as soluble proteins or aggregates in inclusion bodies, and both forms can be purified to homogeneity. In this chapter, we describe an ion-exchange chromatography-based protocol that we have developed involving the use of two anion-exchange columns to purify untagged human AAT from E. coli. We also outline methods that can be used to determine the inhibitory activity of both AAT in cell lysates and purified AAT. Our protocol for the purification of bacterially expressed AAT yields pure and active protein at 6-7 mg/l culture. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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Unknown | 15 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Bachelor | 3 | 20% |
Other | 2 | 13% |
Researcher | 2 | 13% |
Professor | 1 | 7% |
Student > Doctoral Student | 1 | 7% |
Other | 2 | 13% |
Unknown | 4 | 27% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 3 | 20% |
Pharmacology, Toxicology and Pharmaceutical Science | 2 | 13% |
Agricultural and Biological Sciences | 2 | 13% |
Medicine and Dentistry | 1 | 7% |
Neuroscience | 1 | 7% |
Other | 0 | 0% |
Unknown | 6 | 40% |