how can I overcome inclusion bodies formation during IPTG induction when I want to express a protein with disulfide?
have a look at https://www.researchgate.net/post/Avoiding-inclusion-bodies-formation-during-IPTG-induction/1
There are several ways you can try:
1. Use lower IPTG concentration (such as 0.1 mM of IPTG final) and lower temperature (such as 16 -18 C for 24 hours) induction conditions.
2. Use fusion tags such as MBP, GST, Trx to increase the solubility of expressed protein.
3. Use E. coli strain such as Origami B(DE3) or SHuffle Express as a host strain to express your protein containing several disulfide bonds.
Dear Hossein Changaei
IB formation could be due to several different reason (eg. protein is toxic, protein is hydrofobic, protein is unfoklded)
if you thibk that the IB formation is due to the fact that disulfides are not correclty formed (which is possible in cytoplasmic expression in E.coli, since the E.coli cytoplasm is reducing) you can try to use the following different approaches to promote the formation of the S-S bonds
1) Use of E.coli strains (as Origami or T7 shuffle) which lack enzimes as Glustaredoxin involved in S-S reduction and promote S-S bond formation in cytoplasm
2) Add to you protien a signal peptide (eg pelB or OmpA leader peptides) for protein expression in the periplasm which iis more oxidating enviroment and purify the protein from it, by performing selective proten extration using osmotick shock.
good luck
manuele
Generally inclusion bodies can be reduced by:
- Inducing at lower cell densities (A600 = 0.5)
- Inducing for a shorter period of time
- Inducing using a lower concentration of the inducing agent (e.g., 0.1 mM IPTG)
- Lowering the temperature to between 20°C and 30°C
https://lab.plygenind.com/how-to-prevent-inclusion-bodies-formation-in-protein-expression
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