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wise to do an enzyme kinetic assay with unpurified enzyme?

enzyme kinetics assay unpurified enzyme

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11 replies to this topic

#1 petenl2004

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Posted 28 June 2013 - 12:54 AM

Hi all,

I'm new in protein work. I have some questions to ask:

First, this is my situation. I cloned the target gene Susy taken from tomato under the control of a promotor very active with glucose, and expressed it in a yeast host. This Susy gene can break down sucrose into UDP-Glucose and fructose. This yeast host also has the gene (Suc) that break down sucrose. However, we grow the transformed yeasts with glucose, and assume that the our target gene Susy is overexpressed, rather the endogenous one (Suc) from the host.

I had the crude protein extracts from the transformed yeasts. In my lab, we don't have facilities to purify the protein, thus I only used Amicon centrifugal filter column to purify and concentrate the crude yeast extract. Then, I did some assay to measure the enzyme activity of this enzyme and saw that the activity (initial velocity Vo) was quite high compared to the negative control samples (extracts from yeast without expression plasmid, or with the empty expression plasmid).
Now I want to go on with the kinetic assay of this enzyme. However, as I saw in the literature, most of them use purified enzyme for the kinetic assay. So, I am hesitating to go on with my enzyme extracts....

Please advice me what to do here. I really appreciate it.
Cheers,
Peter

#2 mdfenko

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Posted 28 June 2013 - 04:54 AM

you can perform preliminary kinetics with partially purified proteins (keep in mind that other components of the mixture may effect the kinetics).

you say you don't have the facilities to purify the protein. what do you have? did you tag the clone?

you can enhance the protein by fractionation with ammonium sulfate and/or acetone.

do you have hplc, fplc or classical ion exchange, hydrophobic interaction or gel filtration chromatography available to you?

Edited by mdfenko, 28 June 2013 - 04:55 AM.

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#3 petenl2004

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Posted 30 June 2013 - 11:29 PM

Hi Mdfenko,

Thank you very much for your advice.

About the clone, I followed a method from a paper in which their clone did not have any tags. Because of that, they had to use quite some steps to purify their recombinant enzymes.

About the facilities, unfortunately my group does not have those fplc or classical ion exhange, etc. because most of people in my group only works with DNA.
So, the best we can do is to buy the disposable columns...Other option is that I will contact other research groups in my university to borrow their facilities for enzyme purification, it's just not very close to my lab and they are often used by their own groups...

#4 mdfenko

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Posted 01 July 2013 - 07:59 AM

if you are going to do this often then you should buy the supplies you need (you don't need hplc or fplc, you can set up classical lc columns, they just take longer to run). if this is a one-time then you should borrow from one of the other labs.
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#5 petenl2004

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Posted 02 July 2013 - 01:59 AM

Hi mdfenko,

Thank you very much for the advice. I'm looking for the alternatives, if it does not work out, 'll try to persuade my PI to purchase the classical lc columns like you mentioned.

Cheers!

#6 labtastic

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Posted 07 July 2013 - 03:16 PM

Do you have a specific reason not to put a his-tag on your protein?

If not, a single PCR, digestion + ligation can do the job (depending on plasmid). Ni-NTA resin is cheap and forever reusable (with proper regeneration methods), and imidazole is cheap too. A one step Ni purification followed by some kind of salt exchange will likely get your protein >80% pure in less than a day's work with nothing more than an eppendorf tube, a micro centrifuge, the ultra-centrifugal filters you already have for buffer exchange and concentration, and some quick tips from the folks down campus who work with proteins on a regular basis. This can calm most all fears of interfering kinetic artifacts from doing the experiments in cell extracts that can't be accounted for with proper controls.

#7 Inmost sun

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Posted 19 July 2013 - 09:34 AM

check the possibilities to block antagonizing enzymes with inhibitors...

#8 petenl2004

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Posted 06 August 2013 - 06:43 AM

Do you have a specific reason not to put a his-tag on your protein?

If not, a single PCR, digestion + ligation can do the job (depending on plasmid). Ni-NTA resin is cheap and forever reusable (with proper regeneration methods), and imidazole is cheap too. A one step Ni purification followed by some kind of salt exchange will likely get your protein >80% pure in less than a day's work with nothing more than an eppendorf tube, a micro centrifuge, the ultra-centrifugal filters you already have for buffer exchange and concentration, and some quick tips from the folks down campus who work with proteins on a regular basis. This can calm most all fears of interfering kinetic artifacts from doing the experiments in cell extracts that can't be accounted for with proper controls.

Hi Labtastic, 

 

Thank you for the advice. Indeed, it would be wise to have a his-tag on my protein. But I blindly followed a paper that worked on similar gene that I'm working on, and did not take into account that my research group does not have those purification facilities that that paper used. The thing is I have quite many constructs to build, it took me quite some times to have them in the expression host. If I started again building new ones with the his-tag, I don't know if my supervisor will let me do it...But I will consider this!



#9 petenl2004

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Posted 06 August 2013 - 06:45 AM

check the possibilities to block antagonizing enzymes with inhibitors...

Thanks Inmost Sun, I did take this into account. But the assay is still very unstable...I guess purifying the extract is the only solution...



#10 HOYAJM

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Posted 19 August 2013 - 10:15 AM

You can not use unpurified enzymes for kinetics. One of the most crucial aspects of an kinetic assay for an enzyme is [E], or enzyme concentration. You will be only guessing what the concentration of your unpurified enzyme is. Thus, your kcat value would be way off and not reliable.



#11 mdfenko

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Posted 19 August 2013 - 08:32 PM

You can not use unpurified enzymes for kinetics. One of the most crucial aspects of an kinetic assay for an enzyme is [E], or enzyme concentration. You will be only guessing what the concentration of your unpurified enzyme is. Thus, your kcat value would be way off and not reliable.

as i pointed out in an earlier post, you can do preliminary work on impure enzyme. kcat is not the only kinetic parameter that may be determined (substrate saturation, inhibitor kinetics, etc). also, you may be able to determine percent of total for your protein of interest at any stage of purification so you can make a better guess of the enzyme concentration.

 

you would, of course, want to perform kinetics with purified enzyme after.


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#12 HOYAJM

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Posted 22 August 2013 - 12:26 PM

 

You can not use unpurified enzymes for kinetics. One of the most crucial aspects of an kinetic assay for an enzyme is [E], or enzyme concentration. You will be only guessing what the concentration of your unpurified enzyme is. Thus, your kcat value would be way off and not reliable.

as i pointed out in an earlier post, you can do preliminary work on impure enzyme. kcat is not the only kinetic parameter that may be determined (substrate saturation, inhibitor kinetics, etc). also, you may be able to determine percent of total for your protein of interest at any stage of purification so you can make a better guess of the enzyme concentration.

 

you would, of course, want to perform kinetics with purified enzyme after.

 

Yes you did. I missed that. All of my enzyme kinetics have been with FRET peptides ($750 for a few mg's) and at that price, I just never bothered until I had pure enzyme. But, if your assays are cheap, then I guess it doesnt hurt to use a crude prep for a trial.







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