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Help with FPLC AIEX/CIEX: Keeping E. coli Protein Separated/Buffer Selection

FPLCAIEX CIEX colicin Ecoli

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#1 Mio Sison

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Posted 10 April 2016 - 06:56 PM

I am purifying a native bacteriocin protein from an induced E. coli lysate.

 

After following a protocol from a paper for almost 6 months, my lack of success in purification was fixed by actually contacting the author. However, I learned that the Q sepharose AIEX column in the paper was chosen because it was applicable to many other projects, and not optimized for the protein I am trying to purify.

 

I am using a Bio-Rad NGC Quest (with additional column switching module)

My protein has a pI of 9.69, 42kDa.

 

I am using preliminary industrial methods for purification, using 2L culture, inducing, centrifuging, and collecting the supernatant containing my protein. This was then concentrated and buffer exchanged into 10mM Tris-Cl pH 8 by tangential flow 30kDa cutoff cassette. The paper used 10mM Tris-Cl buffer pH 8 and +1M NaCl Elution buffer to equilibrate a Q column. According to this information, the protein's exposure to a lower buffer pH makes it positively charged and able to bind to a CIEX column. However, the protein did actually bind to the Q column (along with many contaminants) but was active in a few fractions determined by drop test activity assay. SDS-PAGE showed my protein (in a low amount) with a great deal of bands in the eluent.

 

Realizing that the paper was wrong, I buffer exchanged the sample (by ultrafiltration) I had (which was in 10mM Tris-Cl pH 8) into 25mM sodium phosphate pH 8 and prepared the same buffers (25mM sodium phosphate pH 8) for use on a CIEX 1mL HiTrap SP XL, but everything came out in the flowthrough. Did this not work because my initial buffer was not sodium phosphate? What buffer should I use for my E coli protein?

 

I suspect that the protein is binding to another one so I am thinking about supplementing the buffer with EDTA, DTT, TX-100, or NaCl to keep them separated. Would the supplemented buffer be okay to run on the FPLC? Keep in mind my induction method does not require me to lyse any cells**

 

I am hoping to find a proper buffer to use before just supplementing the one I use currently because I do not want to waste 2L cultures just trying different buffers. Hopefully someone here can help!



#2 mdfenko

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Posted 11 April 2016 - 04:06 AM

why did you change buffers? tris is a great buffer whereas phosphate is at its buffering limit at pH 8.

 

what else is in the buffer?

 

i know i give this link a lot but, if you don't already have it, get the ion exchange handbook download from the ge lifesciences website:

 

http://www.gelifesci...11787&langId=-1


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#3 Mio Sison

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Posted 11 April 2016 - 05:42 PM

I changed buffers because tris was not recommended for CIEX in the handbook/manual, however I have seen it used in literature. There is nothing else in the buffer besides the tris since I used Tris-Cl and Tris-Base to pH the buffer to 8. The same goes for the sodium phosphate: used mono/dibasic to pH. 

 

I do have the handbook and have gone through it many times; it should bind to a cation exchanger based on its pI, but since it isn't, it must be binding to another protein in the debris. The handbook does not mention using buffer supplementation besides cell lysis. I believe I saw articles on BioForum that did confirm that glycerol, triton-X 100, etc. were able to be used on the column.



#4 mdfenko

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Posted 12 April 2016 - 04:02 AM

you may be able to determine if you are looking at a protein complex by running on sec.

 

edta will only help if a metal ion is involved.

 

dtt may help by maintaining the sulfhydryls, but, don't go to high in concentration or it may dissociate internal disulfides necessary for proper conformation.

 

i don't like to use detergents during chromatography if they can be avoided.

 

is the protein stable at physiological pH? if so, then you can lower the pH a little to increase the charge on the protein.

 

have you monitored the protein content at all stages (eg concentrate and effluent from tangential flow and ultrafiltration)?

 

is your protein globular or filamentous? if it's filamentous then 30 kDa cutoff may too high.


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#5 Mio Sison

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Posted 12 April 2016 - 07:22 AM

The gels I have run showed many bands (15-20) in the eluent fractions, all in very close proximity to each other, will SEC be okay to use with that many proteins? I have run SEC (once, I am a senior in undergrad) in the past and it was not very successful. May have to try it again anyways.

 

 

I had planned to use 1mM DTT, I assume that would be little enough.

 

Is 0.1% TritonX a low enough amount for use on a column.

I believe the protein is stable at physiological pH (this protein is a naturally occuring Ecoli protein but expressed so that it is produced significantly more when induced). I have monitored the TOTAL protein content (since I have yet to purify/isolate it), and at this point I am not losing much from TFF to the eluent fractions (About 0.1mg). 

 

The protein itself has a single filamentous domain and single globular domain. I have checked the filtrate in the TFF and 30kDa concentrators and my target was not there.



#6 mdfenko

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Posted 13 April 2016 - 04:04 AM

we routinely use 2 mM dtt to maintain sulfhydryls without breaking up tertiary and quartenary structures.

 

0.1% triton (and for most other detergents) is pretty much the standard concentration. i don't know if this will disrupt any protein-protein interactions but probably won't.

 

either or both of these treatments may not solve your problem. pH adjustment is more likely to work. however, you should scout the proper conditions to enhance binding and elution.

 

fyi, we've also come across a similar situation where a protein has bound, counter-intuitively, to the opposite ion exchanger than expected at the pH of operation. we just went with it.

 

you don't need to purify with sec to determine the native size of the protein. collect fractions (the smaller, the more accurate), find the peak of your protein (by page) and compare the volume to the peak against a standard curve.

 

keep in mind that just because you see a large number of proteins close in size by sds-page doesn't mean that they are that close natively.


Edited by mdfenko, 13 April 2016 - 04:04 AM.

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#7 Mio Sison

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Posted 13 April 2016 - 04:12 PM

mdfenko, thanks so much for your help!

 

I wish we could just scout optimal binding conditions but the system we have is very basic. It does not have a buffer prep function or sample injector to automate any of our processes. Have to manually prepare every buffer. Desalting with a column is an issue too, seems like our Bio-Gel P-6 has an air bubble at the top and I am not sure if it's okay to use. Been using the ultrafiltration concentrators since.

 

I prepared supplemented buffers today (and those with just minimal salt; my original-tris-cl and 120mM nacl and my new sodium phosphate + 120mM NaCl) and buffer exchanged my supernatant with them. 

 

Im very happy I was not the only person that has just "run with it" when I got things bound to the opposite column; Im not crazy!

 

But hopefully when I run the lysate I will get enough separation for further purification on the SEC. Ill definitely be posting what I find out ASAP



#8 mdfenko

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Posted 14 April 2016 - 07:11 AM

you can scout in a test tube if you have some sp-bound ion exchanger. it's tedious but effective.

 

120 mM salt may be too much when you're having trouble with binding.

 

you should be able to clear the air bubble in your p-6 column. if it's a fplc or hplc column then you can reverse the flow and run it slowly until the bubble exits (assuming a filter or support at the top of the column). this will also decompress the packing.

 

most fplc columns will allow you to remove the top and upper support (filter). then add a little buffer, stir the top, allow it to settle, replace the filter, put the top back on and reattach to the system.

 

you still might be crazybiggrin.png


talent does what it can
genius does what it must
i used to do what i got paid to do






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