GST fusion protein purification

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bsengez
bsengez's picture
GST fusion protein purification

Anyone using B-PER GST fusion protein purification kit?
I performed purification by using this kit. I`m using pGEX2T vector. My protein amount is really low; thus, I have several questions:
1. I`m using E.coli. I began procedure when the culture reached OD600=2. Because it is written to start with OD600=1,5-3 in manual. In most protocols, it is given that OD600=0,5 is suitable. Does it help me if I reduce it to OD600=0,5?
2. Can I add DTT, urea, PMSF or pepstatin to B-PER reagent? I only added 1 tablet EDTA free protease inhibitor coctail. Do you recomend to add these agents with inhibitor coctail to reagent?
3. How can I understand that my fusion protein is insoluble? All steps were easily performed, however the yield is very low per column.
4. Can I add lysozyme to B-PER reagent?
5. Is it necessary to add DNAse or RNAse even if you don`t get viscous sample?
6. Some protocols suggest to grow cells in 20-30 C instead of 37 to increase the soluble protein level. Are you agree with it?
Any suggestion is appreciated.

Ivan Delgado
Ivan Delgado's picture
 

 
Hi bsengez,
Here are some answers to your questions: 
1. I can definitely help you if you use a culture with an OD600 of 0.5 versus one at 2. The reason for this is that cells at 0.5 are alive, healthy, and actively dividing, while cells at 2 are for the most part stationary, dying, and not necessarily very active at making your protein of interest. In other words, at 0.5 theoretically you should get more protein per bacteria compared to using a culture at 2.
2. If you are concerned about protein degradation you can alway add other proteinase inhibitors like PMSF. Adding denaturing agents like DTT or urea may not be the best idea, but I have never heard anything negative about doing this. Alternatively if you are not already doing this you should clone your GST-fusion construct into proteinase-deficient E. coli strains like BL21 DE3
3. There are protocols to solubilize insoluble proteins. If you think this is the problem you may want to go through one of these to test it.
4. I do not see why you wouldn't be able to add lysozyme to B-PER, although that sounds like overkill to me. 
5. No. This should not be a problem.
6. Growing your cells at a lower temperature (20-30oC) can definitely help. This is because when cell grow very fast (37oC) they may also synthesize their proteins very fast, which can lead to protein aggregation. By slowing things down you create a more relaxed environment that in theory will lead to a reduction in protein aggregation.
Good luck

kjosephs
kjosephs's picture
Can you tell us a little

Can you tell us a little about the protein you are expressing like organism, molecular weight, protein class, etc?  What host strain are you expressing in?
Some of these issues are covered in the BPER product literature:
www.piercenet.com/files/0736as8.pdf
Here are a few additional thoughts....
For some proteins whose function is toxic or proteins that express at very low levels it is not uncommon to try inducing at higher cell densities, like OD=2.  The idea is that if the expression host is only going to be able to make very low levels of a protein that you might as well try to increase the number of cells.  Almost every variable in protein expression can be optimized for a particular protein and OD for induction is just one of them.  However, for the majority of protiens inducing in mid-log phase is optimum for the reasons that Ivan gave, and inducing later is never goint to improve your yeild more than a few fold.  It sounds like you are have bigger problems.
I have used BPER with 5mM  BME rather than DTT and it worked fine.  BPER is some sort of proprietary detergent that makes holes in cell membranes and so it is not sensitive to reducing agents, Lyszyme, DNAse, protease inhibitors etc.  I imagine that adding a lot of salt like 8M Urea or an additional detergent might affect BPER lysis, but it's pretty compatible with most things you would want near your protein. 
Lysozyme improves the release of  proteins larger than 70KD that do not leak through the holes created by BPER very efficiently becuase they are too big.  GST is 26KD so if your protein is 40 KD or bigger or is a dimer, trimer, etc then this becomes important.  Also this is important for inclusion body isolation for the same reasons.
Growing your cells at a lower temperature (15-25C) is a good idea.  This is especially true if your GST fusion is partitioning in the insoluble fraction, for the reasons that Ivan gave.  Lower temps and low IPTG (I use 0.1 mM a lot) are good ways to slow down expression and improve yeild of soluble protein.

selenahill21
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