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recombinant enzyme activity

recombinant enzyme activity - Recombinant Protein Forum

recombinant enzyme activity - Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


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Old 03-15-2012, 09:17 PM
Pipette Filler
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Question recombinant enzyme activity



Hi, I am interested in an enzyme in a G(+) bacteria. It natually sits on cell membrane.

I have cloned it in E.coli. But the recombinant protein didn't have enzyme activity (I checked it by loading E coli on zymogram).

My question is: how to make a recombinant enzyme have activity? Does the procedure differs a lot for different enzymes, or there is a universal procedure?

Thanks very much! Look forward to hear from you!
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Old 04-04-2012, 11:36 PM
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Default Re: recombinant enzyme activity

did the protein partition into the soluble or insoluble fraction of bacteria lysate ?

G.
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Old 04-05-2012, 01:52 AM
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Default Re: recombinant enzyme activity

there are just so many thing can go wrong here.
1. Are you sure the protein you clone is the correct one?
2. Did it fold correctly?
3. Many more.

But to immediately answer your question, it depend on your enzyme. Remember enzyme have half life so don't put it at rt without addictive or stuff you use to stabilize your enzyme too long.

Did you frozen it? Most time but generally depend, some enzyme does lose its activity especially after freeze and thaw or frozen.

It would be great if we know what enzyme is it.
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Old 04-05-2012, 02:08 PM
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Default Re: recombinant enzyme activity

Hi! Thanks! I read that some proteins may be in the inclusion body. Do you know how to test it?

For my case, I l didn't check the solubel or insoluble fraction. I loaded the whole bacteria (lysed or not lysed) to zymogram. They don't show activity. I am thinking if they are inside the inclusion body, they cannot be shown in the zymogram.


Quote:
Originally Posted by gturcatel View Post
did the protein partition into the soluble or insoluble fraction of bacteria lysate ?

G.
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Old 04-05-2012, 02:11 PM
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Default Re: recombinant enzyme activity

Thanks a lot for your response!

Yes, I am checking if it is the correct protein, also I am now reading on protein refolding. But I cannot think of any other reason. Would you please give me some expample of "Many more"? So that I could consider it when I design the experiment?

Thanks very much!

Quote:
Originally Posted by butters View Post
there are just so many thing can go wrong here.
1. Are you sure the protein you clone is the correct one?
2. Did it fold correctly?
3. Many more.

But to immediately answer your question, it depend on your enzyme. Remember enzyme have half life so don't put it at rt without addictive or stuff you use to stabilize your enzyme too long.

Did you frozen it? Most time but generally depend, some enzyme does lose its activity especially after freeze and thaw or frozen.

It would be great if we know what enzyme is it.
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Old 07-28-2012, 05:00 AM
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Default Re: recombinant enzyme activity

NICE information thanks
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