Chapter title |
Expression and Purification of Site-Specifically Lysine-Acetylated and Natively-Folded Proteins for Biophysical Investigations
|
---|---|
Chapter number | 11 |
Book title |
Noncanonical Amino Acids
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7574-7_11 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7573-0, 978-1-4939-7574-7
|
Authors |
Michael Lammers |
Abstract |
N-(ε)-lysine-acetylation (short: lysine-acetylation) is a dynamic and powerful posttranslational modification to regulate protein function. Mutational approaches are often poor to access the real mechanistic impact of lysine-acetylation at the molecular level. Therefore, the ability to site-specifically incorporate N-(ε)-acetyl-L-lysine (short: AcK) into proteins dramatically increased our understanding how lysine-acetylation regulates protein function by using diverse molecular mechanisms going far beyond neutralizing a positive charge at the lysine-side chain. Genetically encoding AcK is a powerful way to introduce AcK into proteins, resulting in homogenously, quantitatively, and site-specifically lysine-acetylated proteins. Thereby, lysine-acetylated proteins can be produced in their natively-folded state in a high quality and in a yield sufficient to perform biophysical studies, including X-ray crystallography. This protocol describes the expression and purification of site-specifically lysine-acetylated proteins in Escherichia coli using the genetic-code expansion concept (GCEC) and subsequent steps to assess the successful incorporation of AcK by immunoblotting and mass-spectrometry. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 11 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 3 | 27% |
Student > Bachelor | 3 | 27% |
Professor > Associate Professor | 1 | 9% |
Student > Master | 1 | 9% |
Unknown | 3 | 27% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 7 | 64% |
Nursing and Health Professions | 1 | 9% |
Agricultural and Biological Sciences | 1 | 9% |
Chemistry | 1 | 9% |
Unknown | 1 | 9% |