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Throbin cleavage declines with older protein?


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#1 Dr Rob

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Posted 11 August 2009 - 01:16 AM

Hi all, many thanks for your help in advance!

So I'm purifying three GST fusion proteins, from pGEX 4T-1. Have sequenced all three and the constructs are exactly as expected, including the throbin site. There are no other thrombin sites other than between GST and the fusion proteins. I purified the proteins, got an absoute ton for all three constructs, and left them bound to GST beads at 4 degrees in PBS with PMSF and a little tween. I've been optimising the elution and cleavage of these proteins, at first just trying different volumes and concentrations of glutathione or thrombin. At first it was working great, I washed and cleaved about 30ug of protein in 100ul of PBS using 1U of thrombin, and got about 50% cleavage, maybe more, overnight. So I tried a larger volume and noticed that cleavage wasn't as effective. Then recently I tried another volume and I didn't detect any free protein in the S/N. I went back and tried the original conditions where it worked great, and again nothing was in the S/N. I made sure to use different aliquots of the enzyme so its definately not a freeze thaw issue, and my co-worker has had no problems using the enzyme recently either. Whats strange is that when I wash the beads and run them on a gel, I can see just GST, some fusion protein and loads of cleaved product. This would suggest that the cleaved protein was binding back onto either the beads or GST. I'm stumped as it was fine before, and its happening to all three different proteins. I was thinking perhaps since this purified protein is now about a month old that over time it has denatured somewhat and can therefore bind the beads or GST, which is why it used to work ok, but now when its cleaved I can only detect it in the beads when run on a gel. Any ideas? The only thing I'm doing is repurifying all three from ecoli to start with fresh protein, but I don't want the same thing to happen again.

Has anyone had experience of cleaved protein being detected in the beads sample and not the S/N, or of cleavage becoming totally inefficient on older proteins?

Thanks again,

Rob

#2 klinmed

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Posted 13 August 2009 - 10:00 AM

Hi all, many thanks for your help in advance!

So I'm purifying three GST fusion proteins, from pGEX 4T-1. Have sequenced all three and the constructs are exactly as expected, including the throbin site. There are no other thrombin sites other than between GST and the fusion proteins. I purified the proteins, got an absoute ton for all three constructs, and left them bound to GST beads at 4 degrees in PBS with PMSF and a little tween. I've been optimising the elution and cleavage of these proteins, at first just trying different volumes and concentrations of glutathione or thrombin. At first it was working great, I washed and cleaved about 30ug of protein in 100ul of PBS using 1U of thrombin, and got about 50% cleavage, maybe more, overnight. So I tried a larger volume and noticed that cleavage wasn't as effective. Then recently I tried another volume and I didn't detect any free protein in the S/N. I went back and tried the original conditions where it worked great, and again nothing was in the S/N. I made sure to use different aliquots of the enzyme so its definately not a freeze thaw issue, and my co-worker has had no problems using the enzyme recently either. Whats strange is that when I wash the beads and run them on a gel, I can see just GST, some fusion protein and loads of cleaved product. This would suggest that the cleaved protein was binding back onto either the beads or GST. I'm stumped as it was fine before, and its happening to all three different proteins. I was thinking perhaps since this purified protein is now about a month old that over time it has denatured somewhat and can therefore bind the beads or GST, which is why it used to work ok, but now when its cleaved I can only detect it in the beads when run on a gel. Any ideas? The only thing I'm doing is repurifying all three from ecoli to start with fresh protein, but I don't want the same thing to happen again.

Has anyone had experience of cleaved protein being detected in the beads sample and not the S/N, or of cleavage becoming totally inefficient on older proteins?

Thanks again,

Rob

Just a thought:

Does your protein contain -SH or S-S?
On long-term storage of your protein attached to the beads you may get redox shuffling. This could result in covalent S-S bridges between your protein and the GST.

If this is the case, you could try storing the beads-protein in a slightly acidic buffer (say pH 5-6) containing 1mM EDTA (minimize -SH reactions an disulfide exchange). Then wash beads into cleavage buffer just before thrombin treatment.

Hope this helps




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