5 replies to this topic

[Ivanov_br]

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!  

[swanny]

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.

[Ivanov_br]

swanny, on Mar 2 2009, 12:42 AM, said:

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!

[Mars]

swanny, on Mar 1 2009, 08:42 PM, said:

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. ^^

[swanny]

Mars, on Mar 21 2009, 01:00 AM, said:

swanny, on Mar 1 2009, 08:42 PM, said:

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?

[Ivanov_br]

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!!!

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