id	author	title	date	pages	extension	mime	words	sentences	flesch	summary	cache	txt
cord-265453-z6aux01t	Bierig, Tobias	Design, expression, purification and characterization of a YFP-tagged 2019-nCoV spike receptor-binding domain construct	2020-09-29		.txt	text/plain	3438	183	58	The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion. Here, we describe a construct and protocol for the expression and purification of yellow fluorescent protein (YFP) labeled 2019-nCoV spike RBD. The fusion protein, in the vector pcDNA 4/TO, comprises an N-terminal interferon alpha 2 (IFNα2) signal peptide, an eYFP, a FLAG-tag, a human rhinovirus 3C protease cleavage site, the RBD of the 2019-nCoV spike protein and a C-terminal 8x His-tag. Our experiments confirmed that the fusion protein (also after proteolytic removal of YFP) binds the human ACE2 peptidase domain. Ni-NTA IMAC After removal of detached cells by centrifugation, the expression medium containing the secreted fusion protein was supplemented with 1/4 tablet of protease inhibitor (complete EDTA-free protease inhibitor cocktail tablets, Roche Diagnostic GmbH, 45148300) and transferred to a 15 ml Falcon tube containing 200 l of washed, pre-equilibrated Ni-NTA agarose (Qiagen Cat. No.	./cache/cord-265453-z6aux01t.txt	./txt/cord-265453-z6aux01t.txt