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Problems in recombinant protein expression


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5 replies to this topic

#1 vels

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Posted 20 April 2011 - 09:58 AM

Hi all,

I cloned a bacterial surface protein without N-terminal signal peptide in pET15b vector and expressed in BL21 host. After induction at 37 degrees celsius, I just boiled the cell pellet of a positive clone in sample buffer for 8 min, centrifuged for 10 min and loaded onto 12% SDS gel. I could not able to see any expressed protein. Then, I purified the expressed protein after induction at 37 degrees celsius using metal affinity resin specific for His-tag. The purified protein was mixed with sample buffer, boiled for 8 min, centrifuged for 5 min and loaded onto 12% SDS gel. Then also, I could not able to see any expressed protein. Later, instead of IPTG, I used auto-induction kit also. But, there is no change in outcome. What could be the reason and how to get over expression?

#2 Adrian K

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Posted 21 April 2011 - 03:27 AM

Try express using a milder condition: 25 C or below... lesser IPTG concentration, and longer time of expression. See if it helps....
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#3 vels

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Posted 21 April 2011 - 09:31 AM

Thank you and I will try that. Please suggest me any other changes to be made for conditions of expression.

#4 vels

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Posted 03 May 2011 - 10:49 AM

Thank you and I will try that. Please suggest me any other changes to be made for conditions of expression.


I tried overnight expression at 23 degress Celsius. Still, it did not express the protein. Please suggest me in this regard.

#5 pDNA

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Posted 03 May 2011 - 11:28 AM

what about cell growth? ...do you measured OD every hour after induction?

Do your cells stop growing? ...is it possible that the cells die after induction?

At what OD you induce your recombinant culture?

Regards,
p

Edited by pDNA, 03 May 2011 - 11:28 AM.


#6 Adrian K

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Posted 04 May 2011 - 08:47 AM

wait a minute... do you do western blot to confirm the expression?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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