Asset Details
Do you wish to reserve the book?
Screening and large-scale expression of membrane proteins in mammalian cells for structural studies
Hey, we have placed the reservation for you!
By the way, why not check out events that you can attend while you pick your title.
Oops! Something went wrong.
Looks like we were not able to place the reservation. Kindly try again later.
Are you sure you want to remove the book from the shelf?
Screening and large-scale expression of membrane proteins in mammalian cells for structural studies
Do you wish to request the book?
Screening and large-scale expression of membrane proteins in mammalian cells for structural studies
Please be aware that the book you have requested cannot be checked out. If you would like to checkout this book, you can reserve another copy
We have requested the book for you!
Your request is successful and it will be processed during the Library working hours. Please check the status of your request in My Requests.
Oops! Something went wrong.
Looks like we were not able to place your request. Kindly try again later.
Journal Article
Screening and large-scale expression of membrane proteins in mammalian cells for structural studies
2014
Overview
It can be difficult to express large amounts of membrane proteins for structural analysis. Using pEG BacMam it is possible to screen potential candidate proteins as well as to scale up expression in mammalian cells.
Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His
8
–tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI
−
(
N
-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and
Caenorhabditis elegans
glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4–6 weeks.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
/ 631/535
/ Acid Sensing Ion Channels - genetics
/ Acid Sensing Ion Channels - metabolism
/ Animals
/ Caenorhabditis elegans Proteins - genetics
/ Caenorhabditis elegans Proteins - metabolism
/ Chickens
/ Chloride Channels - genetics
/ Chloride Channels - metabolism
/ Cloning
/ Computational Biology/Bioinformatics
/ Green Fluorescent Proteins - genetics
/ Green Fluorescent Proteins - metabolism
/ Humans
/ Mammals
/ Membrane Proteins - genetics
/ Membrane Proteins - metabolism
/ Methods
/ N-Acetylglucosaminyltransferases - metabolism
/ N-Acetylglucosinyldiphosphodolichol N-acetylglucosaminyltransferase
/ Protein Engineering - methods
/ Proteins
/ Protocol
/ Recombinant Proteins - genetics
/ Recombinant Proteins - metabolism
