In situ proteolysis for protein crystallization and structure determination

Structural Genomics Consortium, University of Toronto, 100 College Street, Toronto, Ontario M5G 1L5, Canada.
Nature Methods (Impact Factor: 32.07). 01/2008; 4(12):1019-21. DOI: 10.1038/nmeth1118
Source: PubMed


We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

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Available from: Wael Rabeh, Oct 04, 2015
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    • "To circumvent this we carried out two remedial strategies, namely in situ limited proteolysis and co-crystallization with small ligands. Limited proteolysis is a valuable tool to remove disordered or flexible regions in purified proteins, and can assist in crystal lattice packing [36]. A small-scale treatment of purified hMAT2B with limited amount of trypsin, chymotrypsin or subtilisin generated stable truncated products (Figure 3A, asterisk). "
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    • "Successful strategies have been outlined for the use of proteases to form stable fragments of proteins for crystallization. Recently, proteolysis in situ (addition of trace amounts of trypsin or chymotrypsin) has been reported [30]. In such cases, flexible loops or termini regions—that potentially block crystal contact formation—accessible to the protease are removed. "
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    • "set in the presence of 1:1000 (w/w) (trypsin) (Dong et al, 2007 "
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