Technique for a rapid and efficient purification of the SHV-1 and PSE-2 beta-lactamases

A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 mi) and conditioned in a buffer of low ionic strength (<20 mM) b y 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 pu lse of 1 M NaCl and the eluate directly injected onto a 120-ml Sephacryl S1 00-MR 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-lactamas es. It could easily be scaled up for the treatment of larger samples. (C) 2 000 Elsevier Science B.V. All rights reserved.