Chapter title |
Direct Extraction and Purification of Recombinant Membrane Proteins from Pichia pastoris Protoplasts
|
---|---|
Chapter number | 3 |
Book title |
Membrane Protein Structure and Function Characterization
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-7151-0_3 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7149-7, 978-1-4939-7151-0
|
Authors |
Lucie Hartmann, Estelle Metzger, Noémie Ottelard, Renaud Wagner |
Abstract |
In the past decade, the methylotrophic yeast Pichia pastoris has proved to be one of the most efficient systems for mass production of recombinant eukaryotic membrane proteins (MPs), leading to the crystallization and structure determination for a variety of them. The actual overexpression of functional MPs achieved with this system is, however, often accompanied by the formation of a variable but significant proportion of misfolded and/or aggregated proteins that are co-extracted and co-purified during the purification process. In order to minimize this unwanted phenomenon, we devised a novel procedure in which MPs produced in Pichia pastoris are directly solubilized from whole cells instead of crude membrane preparation. This approach aims at favoring the extraction of correctly folded membrane proteins that have been targeted to the plasma membrane, limiting the solubilization of the misfolded proteins and protein aggregates that are stored in internal membrane compartments. The method described herewith is based on the formation of protoplasts through enzymatic treatment prior to protein solubilization. This chapter details a set of protocols going from yeast cell preparation and protein solubilization to purification using affinity and size exclusion chromatography. |
X Demographics
Geographical breakdown
Country | Count | As % |
---|---|---|
United Kingdom | 2 | 33% |
United States | 1 | 17% |
France | 1 | 17% |
Unknown | 2 | 33% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 4 | 67% |
Scientists | 2 | 33% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 14 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Master | 3 | 21% |
Student > Bachelor | 2 | 14% |
Researcher | 2 | 14% |
Student > Ph. D. Student | 1 | 7% |
Unspecified | 1 | 7% |
Other | 2 | 14% |
Unknown | 3 | 21% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 5 | 36% |
Agricultural and Biological Sciences | 2 | 14% |
Unspecified | 1 | 7% |
Chemical Engineering | 1 | 7% |
Chemistry | 1 | 7% |
Other | 0 | 0% |
Unknown | 4 | 29% |