RESUMO
An approach for re-folding denatured proteins during proteome research by protein folding liquid chromatography (PFLC) is presented. Standard protein, α-chymotrypsin (α-Chy), was selected as a model protein and hydrophobic interaction chromatography was performed as a typical PFLC; the three different α-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 α-Chy was identified and then it was prepared for further investigation. The specific bioactivity of the refolded α-Chy was found to be higher than that of commercial α-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 α-Chy. When the urea concentration reached 6.0 m, the unfolded α-Chy could not be refolded at all.
Assuntos
Quimotripsina/química , Redobramento de Proteína , Ureia/química , Cromatografia Líquida , Interações Hidrofóbicas e Hidrofílicas , Desnaturação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Hydrodynamic chromatography(HDC) and slalom chromatography(SC) called as dynamic liquid chromatography(DLC) were introduced and reviewed, mainly for the recent development of separation principle, theoretical model, and applications. Fifty two
RESUMO
Recombinant human stem cell factor (rhSCF) was produced as an inclusion body by Escherichia coli DH5alpha grown in a 5 l fermentor. Inclusion bodies of rhSCF were purified and solubilized in urea solution, then renatured with simultaneous purification using a high performance hydrophobic interaction chromatographic (HPHIC) squat column. The refolded rhSCF had a purity of 94% and a bioactivity of 1.2 x 10(6 )IU mg(-1)of rhSCF protein. The method described is fast and simple to implement.