| |||||||||
| Login :: Register :: Search forums :: Top Users :: Reagent |
|
Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
|
GST purification probelms
Posted by: Victoria Smyth (IP Hidden, Unregistered user, )
Date: April 25, 2005 05:42AM
Hi there,
I have been expressing both the mouse and human mRNAs of my gene attached to a GST tag. They are approx 70 kDa including the tag and share ~93% amino acid identity. I am encountering much degradation, especially of the mouse protein once induction has started (at 28 degrees celcius). However, my greatest difficulty has been in purifying either protein. They are both present in the centrifuged cell lysate but do not seem to be binding to the columns. This is particularly a problem with the human form, which mostly comes out in the lysate flow-through but also in the first PBS wash, even when using a new column. I am using 10% reduced glutathione in PBS as the elution buffer and finishing off with a high salt wash to regenerate the column. I have, however, been able to express and purify a single domain common to both the mouse and human proteins of approx 30 kDa but not, as yet, the whole protein from either species. Have you any suggestions?
Re: GST purification probelms
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: April 25, 2005 01:07PM
Please provide more information in regrds the following:
What is your expression system is bacterial, yeast, plant, cell culture? Are you expressing the GST at the N or C terminus of the protein? Do you include protease inhibitors in your sample prep? Do you include DNaseI to digest DNA and to lower sample viscosity? Answers of these questions will help us all in the forum to better trouble shoot your problem.
Re: GST purification probelms
Posted by: Vic (IP Hidden, Unregistered user, )
Date: April 26, 2005 08:34AM
The GST fusion proteins are expressed in E coli with an N terminus tag. I add mini complete (Roche) protease inhibitors and Benzonase nuclease is also added as it degrades both DNA and RNA. Lysates are then filtered before applying to column.
Re: GST purification probelms
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: April 26, 2005 12:51PM
Hi,
From my personal experience I have found that DNAse I has a protease activity that typical protease inhibitors are not active against it (may be this the case with Benzonase? check with MERK on this), therefore I would suggest to omit this from your sample prep and shear your DNA by sonication or vortexing with glass beads. I would not worry about RNA as it is less stable especialy with all the internal RNAse activity released from the bacteria. Also, is your protease inhibitor cocktail from Roche EDTA free or with EDTA. As you might know that EDTA will help better inhibit metalloproteases and thus haveing and EDTA with your cocktail may help reduce this issue. I hope this infromation is helpfull, good Luck!
Re: GST purification problems
Posted by: EANON (IP Hidden, Junior member, 19)
Date: July 8, 2005 07:43PM
You say they do not seem to be binding to the columns. Could it be that the GST tag IS binding but your favorite protein is falling off (it is breaking off the GST tag and falling in the flow-through)? A GST tag is somewhat big, you may want to consider using a different tag completely.
Just a thought.
Re: GST purification problems
Posted by: Naveenreddy (IP Hidden, Unregistered user, )
Date: July 10, 2005 01:04PM
i am also having similar kind of problem. my protein of intrest is 55 kDa human protein tagged GST pGEX-4T1 vector. i am geeting good induction, protein is binding to gst beads and once i elute with GSH i am getting good yield . when i am cleaving with thrombin (fused protein bound to gst beads) i am unable to find any protein in thrombin cleavage fraction (on SDSPAGE)after thrombin cleavage once i elute beads with GSH i am getting GST tag in proper region on SDS-PAGE. any suggestions r most welcome
Re: GST purification problems
Posted by: CSORBA TIBOR (IP Hidden, Unregistered user, )
Date: July 12, 2005 03:17AM
pROBABLY YOUR PROTEIN IS KEPT IN SOLUTION BY THE gst TAG, AND AFTER CLEAVAGE PRECIPITATES. tRY TO CEEP IT IN SOLUTION BY ADDING MILD DETERGENTS, OR USE YOUR PROTEIN IN THIS FORM. iT MAY WORK PROPERLY
GST purification problems
Posted by: Bardos Ibolya (IP Hidden, Unregistered user, )
Date: August 1, 2005 01:52AM
Hi ! I have a great problem with the induction of a gex vector. When I induce the bacteria at 37 C all of my protein goes to inclusion body. When I induce them at 22C overnight, the GST fusion splits off. Does anyone know a solution for this problem? Thank you in advance
Re: GST purification problems
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: August 1, 2005 09:52AM
It has to be an error-and-try process for any unknown protein expression: different induction temperature, duration and inducer concentration. it's painful but there is no alternative way. Someone would try to clone genes into different expression vector and transform into different host bacterias at the same time so as to quickly find best way of expression of their protein of interest.
The temperature can go as low as 15c o/n in your particular case to inhibit as much as possible any proteolytic activity in your host bacteria. Hope it helps.
|
We are moving ... Please post to our new community forums
|
|
||||||||