I am working on CX3CL1 after induction with IPTG at 16 degrees for 24 hours and 37 degrees for 4-5 hours. I can see a band on my SDS gel both in the supernatant and pellet. When I do cell lysis and proceed it further for Ni-NTA, I cannot see any elutions. I further done the western blot with anti-His antibody and can see a band in the pellet lane. I would be thankful if anyone could help me why my protein is not coming in soluble form. What can I do?
Dear Karanbir Singh
Just some questions:
Did you express the full lenght gene or did you express a domain?
Did you use a standard BL21(DE3) e.coli strain?
I'm not an expert of CX3CL1 but since it is a mammalian protein is important that you:
1) Remove the N- term signal peptide (predicable with https://services.healthtech.dtu.dk/services/SignalP-5.0/) from the sequence for expression in E.coli since it is a mammallian signal peptide that could not be processed in E.coli
2) Take in consideration that it contain 8 cisteines that may be involved in S-S bond that in E.coli can be assembled only performing periplasmic expression (replace it signal peptide with the OmpA or pelB leader sequence at N-term) or using the Origami or Shuffle strains.
good luck
Manuele
Dear Manuele Martinelli
Thank you for your response.
We are only expressing a domain starting from 25-100 nucleotides in length, and yes, we are using the BL21 E. coli strain only.
Besides the previous answer, if changing strain is not possible, induction and growth conditions must be modified. You need to reduce the production rate of the recombinant protein. Not always works. Reduce inducer concentration and reduce temperature after induction. If this does not work, you need to purify your protein from the inclusion bodies and run a refolding procedure, which is a pain in the ass.
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