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Determining Cofactor for Inactive Enzyme

histag cofactor enyme assay

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#1 james21

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Posted 01 February 2016 - 02:23 PM

     I am having issues restoring activity to an extradiol dioxygenase that I am studying.  It is a recombinant form of a Rhizobium enzyme, expressed with a N-terminal 6xHistag in E. coli BL21 cells.  I suspect that the Histag is somehow responsible for inactivity and I am in the process of cleaving it using thrombin.  

 

     I can't help but thinking that I am overlooking the fact that a cofactor is required by the enzyme.  The same enzyme has been shown to work using the cell lysate and although my goal is to be using purified protein I can't even get the cell lysate to work.  It is my understanding that LB media is supplemented enough to provide minute amounts of possible cofactors for growth.  Additionally I supplemented the LB media with 1mMFeCl2 (ferrous iron binding site on enzyme) and still no activity.  Previous work on this enzyme involved growing in YEM broth, which I can't imagine making a big difference.

 

Would using YEM media over LB make any difference in activity of my enzyme?

 

How does one usually determine which cofactor is required for an enzyme?  Besides looking for conserved metal binding domains is it a good idea to create a cofactor "cocktail" and use that to determine activity?

 

Thank you for any words of advice.



#2 bob1

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Posted 02 February 2016 - 01:22 AM

If the protein is as you say it is, then there is a good chance that the cofactor is Fe2+, however it would be worth trying heme and Fe-S supplementation too. To get correct folding you do need to grow it in the presence of the cofactor, so you should try individual supplementation for common ions such as Zn2+, Mg2+ etc., but it is a lot of work, and may not show anything. 

 

I see from a quick perusal of wikipedia that these proteins are known to have a range of non-standard ions - Cu2+, Ni2+, Mn2+, Co2+ - it would be worth trying these too.



#3 labtastic

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Posted 02 February 2016 - 09:43 AM

Sorry for a stupid question....but do you know your protein is expressing, and is expressing in soluble form, i.e. not in inclusion bodies?

 

While LB should have enough iron to load at least some of your recombinant protein, 1 mM iron is a tremendous amount and almost certainly toxic to growing cells. Drop that concentration at least an order of magnitude to 100uM, or even less.

 

Also, iron is a very tricky co-factor to work with because its redox state is oxygen sensitive. Fe(II) will oxidize to Fe(III) at neutral pH and will form rust, which will precipitate. If you want to supplement your growing cells with excess iron, feed them as iron(iii)-EDTA (you can buy it from Sigma for cheap). This will keep the Fe(III) soluble and bio-available for the cells to take up without the toxicity issues.

 

I don't know much about these dioxygenases, but assuming iron is the co-factor, do you know what oxidation state it needs to be catalytically active? You may be expressing it loaded with Fe(III) but maybe it needs to be reduced to Fe(II) to be active? Perhaps with a reductase, dithionite/methyl viologen, ascorbate, etc?

 

Since you are expressing in BL21, I assume you are using the pET system for expression? Maybe consider using a more tuneable expression system like pBad. It may be advantageous to express smaller amounts of protein per cell so that the cell has the time and resources to load your protein properly in a more controlled manner, rather than hitting it with the hammer that is IPTG/pET system and overloading the endogenous machinery in Ecoli.

 

In the early days to answer these questions, folks would grow up huge cultures of the native bacteria and purify the desired protein natively. A laborious task for sure, but if successful you'll know exactly the native form of the protein and you can adjust your recombinant system thereafter.


Edited by labtastic, 02 February 2016 - 01:04 PM.


#4 james21

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Posted 03 February 2016 - 12:55 PM

The protein appears soluble and my original assay mixture did include ascorbic acid in it.  I had come across a paper where they had mentioned including it for that very reason (reducing iron).  I am expected to receive thrombin in the mail  today and I plan on throwing every possible metal cofactor I can find at that stubborn enzyme..

 

Thanks for all the advice I hope my next post will be good news.



#5 labtastic

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Posted 03 February 2016 - 03:26 PM

When you make your lysates (or purified protein) and you want to test different metal co-factors, I would consider adding some EDTA (20mM?) in an attempt to strip your protein of any metal cofactor any metals that may have been incorrectly incorporated. Desalt (perhaps with a small PD-10 or Nap5 column) to remove the EDTA, then add back in the metal(s) of choice and test for activity. Just a suggestion is all. 



#6 james21

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Posted 04 February 2016 - 10:53 PM

If the protein is as you say it is, then there is a good chance that the cofactor is Fe2+, however it would be worth trying heme and Fe-S supplementation too. To get correct folding you do need to grow it in the presence of the cofactor, so you should try individual supplementation for common ions such as Zn2+, Mg2+ etc., but it is a lot of work, and may not show anything. 

 

I see from a quick perusal of wikipedia that these proteins are known to have a range of non-standard ions - Cu2+, Ni2+, Mn2+, Co2+ - it would be worth trying these too.

 

By Fe-S do you mean Ferrous Sulfate?  The results from testing the enzyme with different metal cofactors showed that Ferrous Sulfate may be important to the reaction.  The results from supplementing the reaction with FeCl2 did not produce similar results.  If you did mean to suggest Ferrous Sulfate by Fe-S, how is it different from other forms of iron?



#7 bob1

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Posted 04 February 2016 - 11:28 PM

I was meaning some supplementation with iron and sulphur, as these seem to be involved in some dioxygenases.







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