Hello everyone,
I'm currently tryning to overexpressed and purified a protein from saccharomyces cerevisiea into BL21 (DE3).
I'm currently using the pRSET plasmid and normal BL21 (DE3)
However, i'm not able to see any overexpression i tried different protocol :
- I changed the BL21 (DE3) and also try the Lemo21 from NEB
- I verified the construct by sequencing (everything seems correct)
- I pick a lot colonies ( around 20 and more) from differents plates
- I used different growth condition and induction condition Growth/Induction 37/20 37/30 24/20 37/16 ...
- I tried different IPTG concentration (1 mM/ 0.5 mM/ 0.2mM)
- To verify the induction I'm taking a sample directly after the induction but I also tried with a sample after sonication to check if I could see the protein but no result.
My second idea was that the overexpression was very small so I'm currently trying (200 mL of culture in TB media to try directly the purification), but I'm not very confident.
One of my issue is that depending of the colony I picked it's that the time to get the OD beetween 0.4 and 0.6 is really variable.
If someone has any idea, I never used this plasmid before. I'm probably doing something incorect but i don't see what.
Best regards
DEar Damien Bretagne
since you are expressing an eucariot protein in a bacterial host, did you check your DNA sequence for presence of RARE codons?
Can you told us some more details about the properties of your protein'
is it a souble protein, a soluble domain of a membrane protein? is it a membrane protein (since you are terstimng the LEMO strain that is designed for membrane proteins)?
Does it contain many cisteines?
Which is its MW?
Since eucariotic protein expression in E.coli could be tricky and you need to desing the clone and protocoll on the basis of the protein properties,
In case or many rare codons, use of a codon optimize DNA construct or ROsetta strain can be a solution:
in case of many cisteines periplasmic expression or Origami strain coull be a solution
In case of low molecular weight o low expression also addiction of N-term fusion tag as MBP, GST, Trx, GB1, Z can be a solution.
best
Manuele
Hi Manuele Martine,
Thanks to taking time to help me. To answer your question.
RARE codons : Base on different analysis tool my CAI is beetween 0.5 and 0.6.
Protein : The protein is BTS1 the Geranylgeranyl pyrophosphate synthase. It should be a soluble protein. There is a paper on it with the crystal structure and no particular problem of expression, purification are reported.
I tested the Lemo 21 because it can be used for difficult protein and i will need it for futher project. It's a protein of 38 kDA with the Histag it's should be around 40-42kDa
Does it contain many cisteines? My GC rapport it's around 31 % wich based on GenScript tool is good.
I understand that I have to adapt the cloning but it's seems not a really difficult protein to express specially according with the paper crystal structure, but I'm not able to see the induction.
Let me know if you need more details
Best regards
Damien
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