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protein expression (temperature issue)


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#1 simlez

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Posted 25 October 2009 - 06:55 AM

Hi,

I am trying to express a protein from pGEX-5X-1 vector. It was found to be in the inclusion body when expressed at 37C and 30C. I would like to express the protein in the soluble form by lowering the temperature. I am thinking of trying the expression at 18C.

I see that usually papers would induce around 18 hours. I would like to know if 5 hours would be enough to produce the protein?
Any suggestions?

Thanks in advance.

#2 swanny

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Posted 25 October 2009 - 05:11 PM

At 18 degrees, an overnight expression would probably be better.

If you're still not having any joy, some people have started using autoinduction media for proteins with lower solubility, because the autoinduction process appears to be gentler than the IPGT induction sledgehammer.
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#3 KAUSHIK THAKKAR

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Posted 26 October 2009 - 01:46 AM

Hi,
I think you can try induction at 18 degrees.
But I would suggest you take hourly samples may be for first 6 hours and then finally after 18 hours.
Check all the samples on SDS PAGE and then decide how much time you should incubate..

regards,
Kaushik

Hi,

I am trying to express a protein from pGEX-5X-1 vector. It was found to be in the inclusion body when expressed at 37C and 30C. I would like to express the protein in the soluble form by lowering the temperature. I am thinking of trying the expression at 18C.

I see that usually papers would induce around 18 hours. I would like to know if 5 hours would be enough to produce the protein?
Any suggestions?

Thanks in advance.



#4 Prep!

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Posted 26 October 2009 - 02:14 AM

a more complicated solution can be adition of a soluble fusion tag!!!
as kaushik says you can do a qualitative test by SDS or if you are also interested in the yield or expression, try developing an HPLC method!!!
Best luck!!!
Support bacteria - They are the only culture some people have!!!
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#5 Vini

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Posted 27 October 2009 - 03:42 AM

a more complicated solution can be adition of a soluble fusion tag!!!
as kaushik says you can do a qualitative test by SDS or if you are also interested in the yield or expression, try developing an HPLC method!!!
Best luck!!!



Man!!!! y bother wid such a complicated approach in d beginning???....hey, smilez....5 hrs of induction is 2 less. just put it 4 around 14-16 hrs n chk.

#6 Prep!

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Posted 27 October 2009 - 03:46 AM

a more complicated solution can be adition of a soluble fusion tag!!!
as kaushik says you can do a qualitative test by SDS or if you are also interested in the yield or expression, try developing an HPLC method!!!
Best luck!!!



Man!!!! y bother wid such a complicated approach in d beginning???....hey, smilez....5 hrs of induction is 2 less. just put it 4 around 14-16 hrs n chk.


lol!!!
:)
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#7 Costa Rica Bienes Raices

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Posted 27 October 2009 - 03:07 PM

Las proteínas Constituyen Alrededor del 50% del peso seco de los tejidos y no existe Proceso biológico alguno que no dependa de la Participación de este tipo de sustancias. PARA MAYOR Tener el control de tus alimentos ES NECESARIO clasificarlos en tu casa.
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http://www.ramrealtors.net

Edited by Costa Rica Bienes Raices, 27 October 2009 - 03:11 PM.


#8 simlez

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Posted 30 October 2009 - 04:02 PM

Thanks all for the replies!

I've tried O/N at 18C... It seems that the protein is still expressed mainly as inclusion bodies.

I also tried 5 hours at 18C. It is enough to express the protein but the yield is much lower and the protein is still expressed as inclusion bodies.

Also, I have another problem. My target protein seems to be very close to host cell protein when I run sds page for my uninduced and induced sample. I have no clue whether is it leaky expression or is it host cell protein.

Any suggestion on how to differentiate them?

Thanks in advance!

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Posted 30 October 2009 - 05:53 PM

Any suggestion on how to differentiate them?

Thanks in advance!


do a western!!!
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#10 simlez

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Posted 30 October 2009 - 06:01 PM

That's the problem.. I think my lab doesn't have western...

is there any other method besides western?

#11 Vini

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Posted 31 October 2009 - 10:43 PM

That's the problem.. I think my lab doesn't have western...

is there any other method besides western?



if u hv mass spec facility, cut the bands , trypsinize and identify on MALDI.
all the best. :)

#12 Vini

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Posted 31 October 2009 - 10:47 PM

That's the problem.. I think my lab doesn't have western...

is there any other method besides western?



if u hv mass spec facility, cut the bands , trypsinize and identify on MALDI.
all the best. :)


further, 'bout the inclusion body prob, u might hv to pull it out by urea denaturation method :) , or maybe try cloning the gene into some other vector like chaperon co-expressing one???

#13 KAUSHIK THAKKAR

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Posted 01 November 2009 - 10:27 PM

Hey,
Is it possible to upload a gel image of the result..?
Also mention the desired protein size and the other conditions.

Perhaps, then it would be easier to identify the problem.

regards,
Kaushik

That's the problem.. I think my lab doesn't have western...

is there any other method besides western?



if u hv mass spec facility, cut the bands , trypsinize and identify on MALDI.
all the best. :lol:


further, 'bout the inclusion body prob, u might hv to pull it out by urea denaturation method :D , or maybe try cloning the gene into some other vector like chaperon co-expressing one???



#14 Prep!

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Posted 01 November 2009 - 10:30 PM

That's the problem.. I think my lab doesn't have western...

is there any other method besides western?



Do an ELISA (if your lab has :D ) or a HPLC (for this u need to have the standard to match the Retension times)!!!
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#15 swanny

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Posted 02 November 2009 - 04:46 PM

That's the problem.. I think my lab doesn't have western...

is there any other method besides western?



if u hv mass spec facility, cut the bands , trypsinize and identify on MALDI.
all the best. :)


further, 'bout the inclusion body prob, u might hv to pull it out by urea denaturation method :( , or maybe try cloning the gene into some other vector like chaperon co-expressing one???

... or clone as a different fusion protein. Try a His tag or GFP fusion
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.




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