pET SUMO: pros and cons? - (Oct/09/2014 )

Hi,

We would like to use pET SUMO protein expression system to produce rNP of a CCHFV. Before starting we would like also to know what is the complications? is there any base length limitation? or any other difficulties?

Thanks

Selma

have you looked at the manual? the link for the pdf is at this lifetechnologies webpage. it should, at least, give you the answer to your question about base length limitations.

yes i looked and the avarage insert size is between 400-700. We want to express three different protein in length 358, 756 and 1449 bp. i think only 1449 bp could be problematic. So i would like to get information from who work with pET SUMO. maybe he/she has already exressed a proteine in that length. Just one hope! :) thank you for answer

We commonly express proteins using a home-made pET28 SUMO fusion expression vector. Works great, and often (though not always) helps with expression. It is our usual go-to vector when expressing new proteins, and if that doesn't work out then we will move to other vectors after that. No limitations specifically in terms of size of protein expressed with a sumo tag (1500bp is no big deal). Only limitation is whether Ecoli can express that particular protein. Just have to test it for yourself. 

Pros: can often help with expression and solubility, can produce virtually native protein (tagless, no extraneous amino acids) after cleavage by ULP-1

Cons: doesn't always help expression (I've seen some cases where it worsened expression, and I've proteins that still went into inclusion bodies with a SUMO tag), uses N-terminal affinity tag (translation truncation products are purified which is not the case with Cterm tags, which makes it not compatible with non-natural amino acid incorporation), non-specific cleavage of the SUMO tag by Ecoli proteins is not uncommon

When it comes to protein expression, you often just have to try a variety of constructs in a variety of vectors. I recently made and test expressed 18 different constructs for a particular protein. Found only one that worked.

labtastic on Thu Oct 9 20:05:02 2014 said:

We commonly express proteins using a home-made pET28 SUMO fusion expression vector. Works great, and often (though not always) helps with expression. It is our usual go-to vector when expressing new proteins, and if that doesn't work out then we will move to other vectors after that. No limitations specifically in terms of size of protein expressed with a sumo tag (1500bp is no big deal). Only limitation is whether Ecoli can express that particular protein. Just have to test it for yourself. 

 

Pros: can often help with expression and solubility, can produce virtually native protein (tagless, no extraneous amino acids) after cleavage by ULP-1

 

Cons: doesn't always help expression (I've seen some cases where it worsened expression, and I've proteins that still went into inclusion bodies with a SUMO tag), uses N-terminal affinity tag (translation truncation products are purified which is not the case with Cterm tags, which makes it not compatible with non-natural amino acid incorporation), non-specific cleavage of the SUMO tag by Ecoli proteins is not uncommon

 

When it comes to protein expression, you often just have to try a variety of constructs in a variety of vectors. I recently made and test expressed 18 different constructs for a particular protein. Found only one that worked.

thanks a lot!