PEDS Advance Access originally published online on December 3, 2004
Protein Engineering Design and Selection 2004 17(11):779-786; doi:10.1093/protein/gzh092
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Negatively charged purification tags for selective anion-exchange recovery
Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden
1 To whom correspondence should be addressed. E-mail: sophia.hober{at}biotech.kth.se
A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple 
-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low -helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.
Received July 6, 2004; revised November 1, 2004; accepted November 22, 2004.
Edited by Lars Baltzer