[en] A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 ml) and conditioned in a buffer of low ionic strength (<20 mM) by filtration through a 53-ml G25 molecular sieve column, is adsorbed on a 1.7-ml ion-exchange (SOURCE) column. The proteins are released by a 10-ml pulse of 1 M NaCl and the eluate directly injected onto a 120-ml Sephacryl S100-HR column. The very low volume of the eluate ensures optimal conditions and resolution for the molecular sieving process. The method is applied as the polishing step in the purification of the SHV-1 and PSE-2 beta-lactamases. It could easily be scaled up for the treatment of larger samples.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Bouillenne, Fabrice ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Matagne, André ; Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
Joris, Bernard ; Université de Liège - ULiège > Département des sciences de la vie > Physiologie et génétique bactériennes - Centre d'ingénierie des protéines
Frère, Jean-Marie ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Language :
English
Title :
Technique for a rapid and efficient purification of the SHV-1 and PSE-2 beta-lactamases.
Publication date :
2000
Journal title :
Journal of Chromatography. B, Biomedical Sciences and Applications