Peptide recuperation after FactorXa cleavage using GST fusion - (Sep/18/2012 )
Iam tryng to express a peptide of 6kDa using a GST fusion in E.coli. After cleavage using FactorXa protease, peptide precipitates with the GST...I have tried using reducing buffers with b-mercapto after cleavage (not before because factorxa is sensible to reducing agents), as the peptide have a Cys residue that may be forming di-sulfur bridges...It clarifies the precipitate, but according to HPLC results it seems that the peptide continues in an aggregated form. HAve you any experience in order to redisolve the aggregated proteins? I think to try with some caotropic agents such us urea...but I have no much experience in this field.
Hola I don´t know if you makes digestion when the fusion protein is bound to resin or after elution. For me It´s better elute the fusion protein and made digestion at low concentration and pH as far as possible of isoelectric point of your protein, (always into the range of activity of protease). After the digestion pass the preparation across the column and recover free peptide from flowthrought. For concentrate it, an ultrafiltration membrane of 3Kd or a ammonium sulphate precipitation followed from a gel filtration could helps you to have your peptide. If this method continues giving aggregates don´t worry to use Urea in all the media after digestion. Buena suerte