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Beads pull down only free GST

pGEX 6p1 GST

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14 replies to this topic

#1 Franzi

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Posted 31 March 2015 - 01:48 AM

Hey you all,

 

i am currently trying to express a protein in a GST-vector (pGEX6p1). I already cloned it successfully into the vector and the right frame etc is proven by sequencing as well as control digest. There are no stop codons within the protein or anything.

I did then the protein expression in Bl21 DE3 cells by using two methods: IPTG induction and auto-induction. For both methods i found out, that the protein is made via a dot-blot using anti-GST as well as anti-fusion protein antibody. Purification was done by batch purification with sepharose 4B beads.

Now here is my problem: when i load the elute from the beads on a SDS gel, there is only GST (26kDA band). No protein-GST band (which would be 70kDa). The protein i only find in the supernatant-without GST at its original size (42kDa).

I use lysis buffer with protease stop, so i don't get how this could happen?! Has someone of you had a similar problem? What i already tried was to do 2 purifications in a row, meaning that i incubated the supernatant/unbound fraction from the first round in a second. But even here i only have the band for GST and not the GST-protein.....

 

Any suggestions?!

Thank you all in advance!

 

 

Franzi



#2 mdfenko

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Posted 31 March 2015 - 04:12 AM

try incubating the beads with sds sample buffer to determine if the protein remains bound after elution.

 

if not then check the flow through and washes for your protein, your protein may not be binding or may be binding too weakly.

 

you say you use sepharose 4b but not what ligand is attached. sepharose alone is for size separations, not affinity. please give a more complete description of your isolation method.


Edited by mdfenko, 31 March 2015 - 04:16 AM.

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#3 Franzi

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Posted 31 March 2015 - 04:25 AM

Thank you for your answer.

 

It is Glutathione Sepharose 4B. I take the cell lysate, incubate it with the beads and elute the proteins from the beads. My problem is not, that my protein remains on the beads, the problem is that from the original protein-GST only the GST is eluted. The protein itself remains in the supernatant/wash etc. The problem seems to be, that the protein is cleaved off from the GST either before or during the batch purification, although there is no protease added.



#4 mdfenko

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Posted 31 March 2015 - 04:28 AM

are you sure the gst is cleaved? it could be masked.

 

there may be endogenous proteases present. do you add inhibitors to the lysis solution?


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#5 Franzi

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Posted 31 March 2015 - 04:37 AM

Yes my lysis buffer contains protease inhibitors.

 

I think it is cleaved because when i load all the samples on a sds gel, i can only see a very big band for GST and not for the GST-protein combination and in the supernatant there is a band for the protein without GST...



#6 mdfenko

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Posted 31 March 2015 - 04:45 AM

do you use a inhibitor cocktail or do you add individual inhibitors?

 

some inhibitors, like pmsf, have a very short half-life in aqueous solution. do you add your inhibitors fresh immediately before use?


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#7 Franzi

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Posted 31 March 2015 - 05:09 AM

For each cell lysis procedure i make new buffer. Add the pmsf (from frozen stock solution) as well as the inhibitor cocktail immediately before usage. It is not individual inhibitors but a cocktail from Roche.



#8 mdfenko

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Posted 31 March 2015 - 05:15 AM

the frozen pmsf stock, is it in aqueous solution or alcohol (100% methanol, ethanol or propanol)?

 

if it's aqueous then it's probably decomposed. try fresh pmsf.


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#9 Franzi

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Posted 31 March 2015 - 05:23 AM

It's in isopropanol



#10 pavoni.ernesto

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Posted 07 April 2015 - 02:12 AM

Hi,

 

your Gluthadione Sepharose 4B is fresh or are you using a regenerated resin?



#11 labtastic

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Posted 07 April 2015 - 10:18 AM

Instead of a dot blot, do a full blot running whole cells immediately after expression. This will tell you whether the cells are producing full length protein. If they are, then you know something is truncating your protein during the purification. If they are not, then construct and expression needs to be optimized. In other words, running a full blot will guide you where to focus your troubleshooting.



#12 Franzi

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Posted 08 April 2015 - 12:00 AM

Hey you, thanks for your answers.

I already loaded the stuff on a sds gel and yes it is the full length protein there. Also sent it to mass spec. The only problem is, that it seems that the GST is cleaved from the protein right during the expression.....

The beads are new, just bought them for these purposes.



#13 mdfenko

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Posted 08 April 2015 - 04:10 AM

it may be a chore but you may want to re-engineer your insert with a different linker that may be less susceptible to cleavage.

 

or you may be able to change your host to one that won't cleave the linker you're using.


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#14 labtastic

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Posted 09 April 2015 - 07:26 AM

Is there any particular reason you have to use a GST tag?  

 

I've found many times that published/lab-standard methods are not always optimized. You'd be surprised that if you ask people why they express and purify their protein a particular way, more often than not it's because "that's what the previous grad student did", or "that's what the paper says to do", or "this what someone told me to do"....rarely is it because "this method has been optimized and it works much better than these other methods, as you can see from this data in my notebook".

 

I've had a lot of luck expressing proteins with cleavable SUMO and GFP tags.

 

If you want to get fancy, you could double tag your protein (eg Nterm His tag, Cterm strep tag) so that you only purify full length, non-proteolyzed protein.



#15 Franzi

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Posted 10 April 2015 - 05:29 AM

Thanks again for your answers.

The main reason i am using GST tag is because we had this construct and it worked in former times. In our lab there is a microbiologist who helped me to optimize the culturing for the auto induction bcause the iptg induction didn't work for me, but also he doesn't know why it is cleaved during expression.

The vector itself/Protein expression with GST tag has a manual or handbook from GE healthcare which is very detailed but also there they don't write anything about it.

Another point of using GST tag is, that i wanted to bind the GST-protein to beads for a pulldown.

Of course i could buy another vector and clone it again but i am not very lucky with cloning biggrin.png







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