Hello everyone,
I´m expressing Fab in E.coli - I´m using for this pETDuet vector (vector for two genes) - Fab part of heavy chain is cloned in 1st MCS, light chain in the 2nd MCS.
This strategy worked well in our lab before for different Fab.
In fact I´m getting insane protein expression (can be seen with comassie gel, for other Fabs in our lab you usually didn´t see the expression without running a Western)!
Problem is, it is mostly the light chain, for some reason it´s expressing way more then the heavy chain and it also is soluble by itself apparently... This vector should give 1:1expression but that´s not the case for me...
I was wondering if you have any ideas how to decrease the light chain expression - these proteins have quite few Cys and form SS bonds so I want to decrease the protein load that the cell has to deal with and also I want to make sure heavy and light chains come nicely together. Right now I´m purifying a lot of light chain dimer :-(
I did go through some other threads and realized that this vector has two T7 promoters and one terminator which means it gives two transcripts - one with both genes on it and one with only the light chain on - this may be the reason why I´m getting more light chain - I have no idea why they designed it this way, does not make any sense to me.
So right now I´m thinking about mutating/deleting the second promoter...
Any help would be great! Or if you have some similar stories :-)
Ondrej
Dear Ondrej ,
unfortunately the T7 promoter is a strong promoter which is strongly transcribed leading to strong expression of proteins in e.coli, pET duet, both the vectors are T7 but it does not guarantee 1:1 expression, moreover you have to realise that small proteins need lesser molecules to be of equi molar strong in comparison to larger molecule. so a solution to your problem can be
1. use a 2 vectors system lnstead of one with plasmids which are compatible with each other to fine tune expression from different plasmids. for this you can search more info on the Novagen website for compatible plasmids.
2. you can mutate your lighter chain promoter to Lac promoter which is not so strong as T7 promoter.
Good luck.
Another option is to generate a polycistronic expression system in which you have one transcript but with both coding sequences and an internal ribosome binding site. In this way you at least have reasonably equal amounts of each message.
In my experiences with these systems, you can often alter the expression levels by changing the order of the genes. I usually see best expression for the gene nearest the promoter and lower levels for the internal ones.
There are many systems available to do this, Song Tan has a beautiful paper describing expression of the Picello complex (I believe) and describes many of the constraints involved in the pST44 system.
Also, I believe there are bacterial strains out there designed to produce a more oxidizing environment to help with expression of proteins that may have disulphide bonds. Try the Origami cells (sold by Novagen).
Best of luck!
Thanks everybody!
So I decided to just delete 2nd T7 promoter - this should give me only one bicistronic transcript (both genes on the same mRNA) and hopefully should give me more equal expression of the two chains. Deletion is done so I´ll be expressing next week - fingers crossed - can´t wait to see how it goes - I´ll keep you all posted!
Thanks again for all the suggestions!
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