Hi,
I am getting difficulty in the expression of pCNF into my protein. I get a wonderful yield while expressing the wild type protein. However, the moment I co-transform the pDule2pCNF into expression host ( to introduce the tRNA for detection of amber codon mutation in the gene) I get almost no expression. I have used the recommended auto induction media composition, 10 mg/50 ml pCNF, pBAD vector and pET21A vector, but no success.
Please help!
Thank you,
Anshuman
Ref: Article Site-Specific Incorporation of Unnatural Amino Acids as Prob...
Hi Anshuman, can you clarify what you mean by you using pBAD and pET21? Do you mean you've tried expressions with pDule2 + pBAD-GFP that didn't work and other expressions with pDule2 + pET21-GFP that also didn't work? Or are you using pDule2 + pBAD + pET21 in the same expression? I ask because the latter won't work since pBAD and pET21 have the same origin of replication (and same antibiotic resistance I believe).
My suggestion is to try incorporating p-azido-phenylalanine as a control. The pCNF synthetase puts this amino acid in much better than pCNF. This would be a good control to test whether the amber suppression system itself is working in your hands, or if the issue is specific to the pCNF amino acid.
Hello Richard, thank you for replying. I have added my gene-of-interest (GOI) to the pBAD-LIC-cloning vector 8A (that has an RBS), pBAD-AmberMutant-GOI which did not express even at 0.2 % L-arabinose induction in BL21-AI as well as TOP10 cells in the presence of 10 mg/ 50 ml pDule2-pCNF for 24 hr incubation at 37 C shaking 200 RPM. However in the absence of pDule2-pCNF, the pBAD-WildType-GOI expresses somewhat in a detectable quantity. I have also tried the expression of my pET21A-WildType-GOI, which comes beautifully well. However, no expression is seen with the pET21A-AmberMutant-GOI with pDule2-pCNF in the presence of 1 mM IPTG and 10 mg/50 ml pCNF in BL21-DE3 cells, also with BL21-AI cells (added 0.2 % L-arabinose ). I don't have a pBAD-GFP control, so i couldn't try a positive control.
Hi Anshuman, sounds like your pET21 system is working well for wild-type. This is a good place to start. It is puzzling why you dont' get amber suppression with your TAG mutant. Keep in mind that not all sites on a protein are permissive for a non-canonical amino acid, so it's possible that changing the TAG site location might be sufficient to get expression.
With that said though, I strongly encourage you to get the pET28-sfGFP wt and 150TAG controls from our addgene site (plasmids #85492 and #85493). It is so much easier to troubleshoot the suppression system with GFP since you can monitor protein expression by cell fluorescence rather than having to do purifications every time. It is most valuable to convince yourself the pCNF system is working in your hands with an ideal protein (sfGFP) at an ideal site (150) before moving into unknown territory with your GOI. Hope that helps.
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