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Thrombin cleaved GST-fusion protein- multiple bands

GST fusion protein

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#1 keyspan

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Posted 21 May 2015 - 07:41 AM

Hi guys,
I am trying to purify my protein of interest of about 72KDa from GST-fusion tag using thrombin to cleave my protein from the glutathione sepharose 4B membrane.

When I cut the GST-fusion protein bound to the column with glutathione sepharose 4B membrane with thrombin, the resulting elution contains a band at the correct size (72 kDa) followed by many smaller bands. 

I also tried to cleave the eluted GST-fusion protein. In this case I see on SDS-page three bands corresponding to GST tag, thrombin and my protein.

I am using Glycerol,Triton, DTT, and the 25X complete cocktail to inhibit protease activity but with seemingly little success. 

The lysis is done using a French press. 

I am hoping to do immunization assay with my protein and need a very pure sample to do so.

If you have any suggestions I would be very appreciative.

I hope in your answer as soon as possible
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#2 labtastic

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Posted 21 May 2015 - 11:11 AM

Sounds like thrombin is cleaving more than it should?

 

Maybe your protein of interest has endogenous sequences (or something close) that thrombin might cut?

 

How are you getting rid of/inactivating the thrombin?

 

Consider doing cleavage for shorter times. Quench with protease inhibitors (eg TLCK), remove thrombin more quickly (with benzamidine sepharose, gel-filtration, etc).

 

Worst case scenario you may likely be able to get rid of those extra bands by ion exchange.

 

On a side note, I haven't used thrombin too much but if memory serves it has internal disulfide bonds in which case you don't have DTT in your reaction do you?



#3 ibmbio

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Posted 23 May 2015 - 06:44 AM

Hello keyspan,

 

As long as you are getting the correct MW band at 72KDa, this is good. The other bands, if one of them represents the GST MW, so this is expected - but you need to optimise the cleavage in a way not to get GST recovered and the most of your protein is cleaved. Try to increase/decrease the incubation time/concentration of the thrombin.

 

To check what the other bands whether or not correlate to the cleavage process, you may consider running SDS-PAGE for the following:

1- sample (A) from the elution without cleavage. Elute using reduced glutathione to see the band of your protein in about 97KDa level. Are there any other bands?

2- sample from the elution with cleavage. Cleave and take sample ( B ), also elute and take sample ( C ) (presumably GST very clear at about 25KDa). Compare samples and conclude.

These steps to check if the thrombin could be an issue. 

 

Regards


Edited by ibmbio, 23 May 2015 - 06:45 AM.





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