The 2 kDa GST Fusion protein was expressed as a in BL21DE3 expression system. After GST purification I want to remove the GST tag using precision protease and separate out the small peptide. I want to use this peptide for structural studies as well as to raise antibody. Is FPLC a good idea for separation ? Can anyone suggest a good idea to do this ?
(Size of GST tag is 26 kDa)
Yes, gel filtration should separate the 2 kDa peptide from GST and from the uncleaved fusion polypeptide and the Precission protease
You bind your fusion on gst beads..you cut overnight on beads with precision , the 2kd peptide is released..you have to get rid of precision protease . Ultrafiltration through amicon 5kd should do the job...alternatively use a tagged precision and after the cut, bind the digest reaction on bead for the affinity tagged protease.
Many possibilities
Hi there,
As Precission protease is itself a GST fusion, it is highly preferable to perform on column digest : both Precission and free GST will remain stuck to the column while peptide will be eluted.
Thanks all. Since the precision protease will have GST tag I can get rit of this through affinity chromatography (with GST beads). Now there will be only 2 kD peptide. Is it good a good idea to run it in gel ? Will I be able to see a band for the 2 KDa one in FPLC ?
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