a problem in protein purification

Jim Woodgett jwoodget at oci.utoronto.ca
Thu Nov 3 11:54:36 EST 1994


In article <9411031605.AA25047 at pobox.upenn.edu>, yluo at POBOX.UPENN.EDU (yuling 
luo) writes:
  
> I am trying to purify a myc-tagged recombinant protein (pI=7.6, 100 
> kd) from the supernatant of baculovirus-infected insect cells. I 
> first enriched the protein on a cation exchange column, which worked 
> out fine. 
> I then loaded the eluate into an anti-myc monoclonal affinity column 
> and eluted the column with 0.1 M glycine pH 2.5. I found that most of 
> the myc-tagged protein bound to the affinity column. But the 
> eluted protein is in precipitated form. It become obvious when I 
> netralize the elute to netral pH with Tris buffer. Then I would just see 
> a big white precipitate forming. I also tried high pH elution buffer. 
> The eluted protein is still precipitated. My questions are 1) could 
> this be the problem of the affinity column itself? (if it is, I could 
> try different ways to purify it) 2) Could this be the protein's 
> problem? (ie, the protein will aggregate in the absence of other 
> proteins, if this is the case, I am hopeless. 3) anyone out there 
> has similar experience? 

Clearly your 100 kDa protein doesn't like being at pH 2.5 (who could blame 
it?).  There are three possibilities you could try.  Try eluting with 
diethylamine (50 mM, freshly pHed to 11.5).  It's possible your protein 
doesn't object so much to alkali, but it's a long shot.  Try eluting with 4M 
MgCl2.  This is chaotropic and has the same potential problems as extreme pH.  
In all cases dilute or neutrilize as quickly as possible (have a buffer in the 
collection tubes).  Finally, synthesize the epitope peptide and use it to 
compete the protein.  This is highly likely to work but is expensive.  It also 
screws up your antibody column as its very difficult to elute all of the 
peptide in a regeneration step.  You can recover the excess peptide that 
elutes with your protein and recycle it.

Why do you need to tag purify in the first place if you are using baculovirus 
expression?  Usually proteins require only 100-500 fold purification from such 
systems.  It might be worth trying conventional purification and following the  
protein by Myc tag immunoblotting.

Jim Woodgett









More information about the Methods mailing list