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GST fusion protein purification


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#1 bsengez

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Posted 20 April 2009 - 11:33 AM

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 welcome.

#2 sudhakar mutyala

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Posted 21 April 2009 - 03:53 AM

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 welcome.


HI
GST tag helps the expressed protein to be in soluble fraction, first of all you need to know how your protein was expressed (Soluble/IB), what is your growing conditions i mean temperature and media, and what is your expression rate. its correct that if you grow your cells at below 25c, most of the protein will be in soluble.

Lyse the cells and try to load the supernatant and solid fractions on a gel so that you will
know in which fraction your protein is there.

and what was the Resin you are using and binding conditions need to optimise the whole process.

If your protein is a enzyme or protein which will degarade easily then maintain cold conditions and add protease tablets. it is not necessary to add DNase and Rnase .(depends on the sample and lysis).

download the GST protein Purification protocol book from GE healthcare website , it will help you to purify the protein.

Yours
Sudhakar m




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