id	author	title	date	pages	extension	mime	words	sentences	flesch	summary	cache	txt
cord-317142-qd61qvch	Muramatsu, Tomonari	Autoprocessing mechanism of severe acute respiratory syndrome coronavirus 3C‐like protease (SARS‐CoV 3CL (pro)) from its polyproteins	2013-03-27		.txt	text/plain	6356	268	56	In vitro 3CL pro autoprocessing system First we developed a SARS 3CL protease autoprocessing system by use of the Escherichia coli cell-free protein synthesis system, with the N-and C-terminal 10 amino acid pro-sequences accompanied by an S tag and a His tag, respectively (Fig. 1A) . Over the course of the cell-free protein synthesis reaction (30°C, 4 h), the N-and C-terminal processing sites of the catalytically active construct (wild-type, WT in Fig. 1A) were completely cleaved by the enzyme's endogenous proteolytic activity, as shown in Fig. 1B ,C (WT). To investigate the activities of the pro-forms of SARS-3CL pro in detail, we developed a 'trans-cleaving assay', using a substrate in which the core region of the 3CL protease was exchanged with GFP (green fluorescent protein) ( Fig. 2A) . We estimated the activity of the enzyme (or pro-enzyme) toward the N-and C-terminal processing sites in the GFP substrate on the basis of the dilution ratio at which 50% cleavage was achieved (Fig. 2C) .	./cache/cord-317142-qd61qvch.txt	./txt/cord-317142-qd61qvch.txt