Projets par an
It has recently been reported that 2-methyl-2,4-pentanediol (MPD) can modulate the protein-binding properties of sodium dodecyl sulfate (SDS), turning it into a non-denaturing detergent. Indeed both alpha (the lysozyme) and beta (the carbonic anhydrase II) soluble enzymes, as well as a beta membrane protein (PagP) have been successfully refolded into their native form by using this amphiphatic alcohol. In order to support the universal character of our MPD-based technique, we have extended its transferability to the Omp2a trimeric membrane porin. The far-UV circular dichroism signature of Omp2a refolded with our original procedure is identical to that obtained by classical techniques, clearly indicating a proper refolding. Moreover, we show that the optimal SDS/MPD ratio for refolding Omp2a is similar to what has been observed for other types of proteins. While the protocol allows refolding at higher protein concentration (up to 4mg/mL) and ionic strength (up to 1M NaCl) than other refolding methods, it is also more efficient at basic pH values and medium temperature (20-40°C). Finally, the key role of the cosolvent was highlighted by a thorough study of the efficiency of MPD analogues, and a high variability was observed, as they can be able or unable to induce refolding at low or high salt concentrations.
Empreinte digitaleExaminer les sujets de recherche de « Towards a universal method for protein refolding: The trimeric beta barrel membrane Omp2a as a test case ». Ensemble, ils forment une empreinte digitale unique.
- 1 Terminé
1/10/10 → 1/10/14
Projet: Projet de thèse