biotin content of F-17 plus TN1 for Streptactin column use

 

Can you purify OneStrep tagged proteins directly from the F-17/TN1 media used for secreted proteins in the Durocher suspension system? I can find no references to biotin content of this combination and this is the first time we will be trying it out.
Thanks
Nick
Posted on 07-May-2012 11:00 CEST

Hi Nick,

if you need a quick solution now, maybe you can try to bind your protein on an ion exchange column first and then elute in biotin free buffer. If you don't know if anion or cation exchange is better just use two columns in tandem so it should be bound by one of them (adjust the pH to a pH at least 1 Unit from the pI of the protein) . You would save time compared to concentrating and dialysing the biotin. I hope that helps.
Best regards

Hüseyin

Posted on 07-May-2012 11:44 CEST

Imre Berger says:

IBA says you can efficiently pre-deplete with neutravidin, then use a Strep column to capture your protein.
We never tried this though so I can not comment on this.

Otherwise - why not TWO tags? One oligo-his (pre-purification) and then Strep. TAP - tag basically.
That we do and it works.

Posted on 07-May-2012 13:21 CEST

Andrea Lages Lino Vala says

Hi Nick,
 
I am not certain about your media per se, but we use FreeStyle from Invitrogen which also has biotin. Invitrogen will not release the data unless you sign a CDA. We are trying to find out the amount by signing this. You might also need to contact the company you obtain the media from and ask them about the bioting content...
 
So far we are not at the point of purifying secreted proteins, so we just have been looking into this problem but haven't really tried any solution.
 
One thing to keep in mind is that depending on which column you are using, they have different biotin capacities. For example Strep-Tactin resins have a biotin capacity of approx 350 nmol/ml sedimented resin. But the new Streptactin High capacity columns from IBA have biotin capacity of around 900 nmol/ml sedimented resin (can't find the exact number).
 
IBA catalog suggests to use 120 mg avidin per mg of contaminating biotin. But this will be a difficult task to do if you don't know the exact amount of biotin in your media! Our plan was to concentrate the media and followed by dialysis into a appropriate buffer using an AKTA crossflow machine. We have it in the lab, but we are still learning how run the AKTA crossflow and how to optimize the process.
 
Also, from our experience with Strep-II tagged proteins, a lot of it will come out in the flow throughout fraction. And the volume of sample you add to the column will affect a lot the binding of the protein to the column. So, you might want to concentrate your media anyway... I have also tried a OneStrep-GFP purification over a StrepTactin high capacity column (IBA) and still saw protein coming out in the flow through and washes fractions. I had approx 30-40 ml to a 5 ml column.
 
Sorry to not be of more help.
 
Best regards,
 
Andrea
Posted on 07-May-2012 13:22 CEST

 

Hi Nick,

 

You can ask invitrogen to produce any media you like with specific components omitted. That is, you could ask for F17 or Freestyle without biotin. If that affects cell growth you can add it back yourself – which may allow you to get away with using less biotin.

 

Adding avidin works well, but becomes expensive for larger cultures. For buffer exchange, we have an akta cross flow and tried it for buffer xchange but it really only works for small scale cultures... for larger cultures hollow fiber concentration/diafiltration is a much better (and faster) option (spectrumlabs for instance).

 

Double tagging is well worth considering as Imre pointed out !

 

 

Cheers

Posted on 07-May-2012 13:23 CEST

 

Sabine Suppmann says:

 

Dear Nick,

Thomas Schmidt, the Strep „expert” recommends to dialyze cell culture media to remove biotin which is always included in media ( and of course not disclosed) or precipitate the secreted protein with Ammoniumsulfate prior to Strep purification.

And also as Andrea mentioned you need a concentrated sample for efficient binding,.

Best

Sabine

 

Posted on 07-May-2012 13:25 CEST

Dear Nick

 

we have identified  of 99 nmol in 50 ml F17 Medium. This means already half a litre of F17 will block a 1 ml conventional Column.
 
Sincerely yours Joop
Posted on 07-May-2012 13:27 CEST

 

 

Hi All,
If my calculation from Joop's (very useful) data  is right then we have approx 2µmol/litre which is roughly 0.5mg. To that we would have to add 60mg of avidin worth about €150 to each litre!!OK luckily we have some data on this protein so I can do a cation exchange to concentrate and buffer exchange. With other proteins we are not so lucky.
Hmmm...OneStrep is beginning to look less attractive/less useful by the minute!
Thank you all for your input-time to get out my S column
Nick

P.S. Why is it so difficult to get this information from e.g. Invitrogen. We only want the concentration of one component and I'm not exactly going to be able to reverse engineer Freestyle F-17 from that info!!
 

 

Posted on 07-May-2012 13:36 CEST

 

Dear Nick,

 

Thank you for the informative call;-)

 

We tested the Freestyle F17 medium for Biotin content and also checked the information with Invitrogen (they say 0,7mg Biotin/ Liter Medium).

 

We measure: 1L FreeStyle F17 medium contains ~0,4mg Biotin =1,4µmol

 

I hope this information helps;-))

 

Best regards

Isabel Schuchardt

Posted on 07-May-2012 17:02 CEST

Hi Nick,

I would suggest something similar than Hussein: use an ion exchange column to bind your protein; you can load them at high flow rate, are cheaper, and you are not afraid to ruin them. After loading, you can wash the column at the maximal conductivity that wash impurities and do not elutes your protein (you eliminate the biotin in this way). Next step is to put your affinity column on tandem with the IEX, and increase the conductivity to elute your protein from the EIX. Wash with same buffer till low OD. Next: disconnect both columns, and finally elute your protein from the affinity column. I used this strategy many times when the affinity resin is very expensive or when there are interferents to the affinity column, or when I have to load high quantities of medium to the column (10 or more liters), so you need a quick capture column.

Mario

Posted on 09-May-2012 0:54 CEST
Displaying 1-10 of 1512Next >