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Recombinant Protein migration


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#1 Ivanov_br

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Posted 27 February 2009 - 05:49 PM

Hello there. As a first timer, a quick but tricky doubt.

I'm trying to express in E. coli a bovine recombinant protein. According to it's sequence, the theoretical MW is 33 KDa, however all papers cite this portein as a 55 KDa.

The questions are:

(1) Is this difference related to glicosilation, phosphorilation on the protein, or related to its aminoacid content?

(2) Probably, the E. coli lineage I'm using wont make pos-translational modifications, so will my protein migrate as 33KDa or 55 Kda?

Thanks in advance and best regard from a shining summer in Brazil! :D

#2 swanny

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Posted 01 March 2009 - 07:42 PM

Size difference of the size you're suspecting would be due to glycosylation, most probably N-glycosylation, because O-glycosylation makes linear sugar chains, rather than more-complex branched sugar chains.

E coli will make the 33 kDa form.
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.

#3 Ivanov_br

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Posted 03 March 2009 - 09:42 AM

Size difference of the size you're suspecting would be due to glycosylation, most probably N-glycosylation, because O-glycosylation makes linear sugar chains, rather than more-complex branched sugar chains.

E coli will make the 33 kDa form.



Thank you for your help. :) At least now I know that a 12% gel will fit!

#4 Mars

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Posted 20 March 2009 - 06:00 AM

Size difference of the size you're suspecting would be due to glycosylation, most probably N-glycosylation, because O-glycosylation makes linear sugar chains, rather than more-complex branched sugar chains.

E coli will make the 33 kDa form.



Hi Swanny, I found one of my proteins has about 33kDa greater molecular weight than expected, the expression was conducted in BL21 strain. I have noticed you are referring to a 33kDa form of glycosylation in E coli previously, could you give me more information about the glycosylation form please? It would be extremely helpful to my experiment. Thanks very much in advance. ^^

#5 swanny

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Posted 22 March 2009 - 07:56 PM

Size difference of the size you're suspecting would be due to glycosylation, most probably N-glycosylation, because O-glycosylation makes linear sugar chains, rather than more-complex branched sugar chains.

E coli will make the 33 kDa form.



Hi Swanny, I found one of my proteins has about 33kDa greater molecular weight than expected, the expression was conducted in BL21 strain. I have noticed you are referring to a 33kDa form of glycosylation in E coli previously, could you give me more information about the glycosylation form please? It would be extremely helpful to my experiment. Thanks very much in advance. ^^

Sorry, I must not have been thinking too clearly, so I didn't write my answer too clearly. E coli won't glycosylate your protein.

What is the expected size of the protein? What did you clone into? Is it a fusion protein?
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.

#6 Ivanov_br

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Posted 20 April 2009 - 07:12 AM

Yes, is a his tagged fusion protein.

Now, after some tests with different E coli types, I found the best conditions. The western blotting with anti histag antibody showed a band with ~34 KDa. (image attached)

Thank you for your help!!!

Attached Thumbnails

  • Wes_O.jpg





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