Refolding of urea-denatured a-chymotrypsin by protein-folding liquid chromatography.

Biomedical chromatography : BMC

PubMedID: 23033217

Congyu K, Wujuan S, Qunzheng Z, Xindu G. Refolding of urea-denatured a-chymotrypsin by protein-folding liquid chromatography. Biomed Chromatogr. 2013;27(4):433-9.
An approach for re-folding denatured proteins during proteome research by protein folding liquid chromatography (PFLC) is presented. Standard protein, a-chymotrypsin (a-Chy), was selected as a model protein and hydrophobic interaction chromatography was performed as a typical PFLC; the three different a-Chy states - urea-denatured (U state), its folded intermediates (M state) and nature state (N state) - were studied during protein folding. Based on the test by matrix-assisted laser desorption/ionization time of flight mass spectrometry and bioactivity, only one stable M state of the a-Chy was identified and then it was prepared for further investigation. The specific bioactivity of the refolded a-Chy was found to be higher than that of commercial a-Chy as the urea concentration in the sample solution ranged from 1.0 to 3.0?m; the highest specific bioactivity at urea concentration was 1.0?m, indicating the possibility for re-folding some proteins that have partially or completely lost their bioactivity, as a dilute urea solution was employed for dissolving the sample. The experiment showed that the peak height of its M state increased with increasing urea concentration, and correspondingly decreased in the amount of the refolded a-Chy. When the urea concentration reached 6.0?m, the unfolded a-Chy could not be refolded at all.