I have cloned a mammalian gene through the gateway cloning (Phase Lamda ) method. The codon optimization was done via IDT tools. The orientation of the insert has been confirmed by Sanger sequencing.
The vector was transformed in E coli BL21 AI cells. recombinant protein expression conditions are :
1. inducing protein expression with 0.2% L-Arabinose, when OD reached 0.5-0.7.
2. Expression temp 37 for 4 hr.
Thankss in advance
Here's a publication that might help you out.
Article Current Protocols in Protein Science
Mammalian proteins are often not expressed well in E. coli for several reasons:
Given these factors, even with codon optimization and confirmation of insert orientation, there can still be challenges in achieving efficient expression of mammalian proteins in E. coli. To overcome these limitations, alternative expression systems such as mammalian cell lines, insect cells, yeast, or in vitro translation systems can be considered, depending on the specific requirements of the protein and downstream applications...
Dear Anil Kumar Gupta
All the reason explained from Noor Rajeeb are right also if each protein has different properties and different history.
In my experience, if you are using a codon optimize gene, in most cases mammalian proteins can be produced in e.coli even if there are some specific proteins, for example protein rich of cisteines or proteins with high mw and of course membrane proteis that are more difficult to be expressed.
In my experience also the addition on N-terminal cleavable (eg adding the TEV site) tags as GST, GB1 or Trx may help to improve the expression on mammalian in E.coli.
Of course the selected protein domain could be also critical.
Did you cloned the entire gene or just a domain?
Did you checked the sequence for the presence of mammalian signal peptide? if yes, did you removed it?
best regards
Manuele
Thank You Manuele for your suggestions. I have cloned single domains of our protein and before cloning, we have checked the presence of signal peptide, cellular location etc.
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