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PLoS One. 2012;7(10):e46147. doi: 10.1371/journal.pone.0046147. Epub 2012 Oct 3.

Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.

Author information

1
Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands.

Abstract

The biophysical stability is an important parameter for protein activity both in vivo and in vitro. Here we propose a method to analyse thermal melting of protein domains in lysates: Fast parallel proteolysis (FASTpp). Combining unfolding by a temperature gradient in a thermal cycler with simultaneous proteolytic cleavage of the unfolded state, we probed stability of single domains in lysates. We validated FASTpp on proteins from 10 kDa to 240 kDa and monitored stabilisation and coupled folding and binding upon interaction with small-molecule ligands. Within a total reaction time of approximately 1 min, we probed subtle stability differences of point mutations with high sensitivity and in agreement with data obtained by intrinsic protein fluorescence. We anticipate a wide range of applications of FASTpp in biomedicine and protein engineering as it requires only standard laboratory equipment.

PMID:
23056252
PMCID:
PMC3463568
DOI:
10.1371/journal.pone.0046147
[Indexed for MEDLINE]
Free PMC Article

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