I have done whole plasmid amplication using XT-20 high fidelity enzyme from merck. After induction with 0.3mM IPTG i did not see any expression of the protein.I am suspecting some mutation has occurred in promoter region. and if it is not then what could be possibilities and solution for this. If anybody does have any suggestion, please give. Thank you in advance.
Hi there,
I guess the wild type protein is expressed in the same system... So 3 trivial possibilities:
1) as you said unwanted mutation(s) has occured which leads to no protein expressed
2) the mutation itself affects the expression of the protein (for some reason it might be that conditions for expression of wt protein are not appropriate for the expression of the mutant version)
3) the mutant protein is expressed but is very unstable during expression/extraction.
First thing to do I think is sequencing of the plasmid (including promoter and terminator) and then if the sequence is correct try to optimize the condition for expression of the mutant (host cell, concentration of inductor, t° and length of induction aso).
Hello prakash,
Some additional information may required to understand your problem exactly.
anyway
1) Was that protein showing any expression before mutagenesis?
if yes, then as you suspected DNA polymerase might have introduced mutations in critical domines or added additional nucleotides while extension process and leads to frame shift
To minimize above issues, find out two unique restriction sites present at ~100bp outward to mutation zone in both ends and Gel-Extract the mutation containing fragment (~200bp) and stitch it to your old vector backbone (which was not come from Mutagenesis process). Then you can sequence verify only mutated fragment instead of total plasmid.
2) On other hand, if u didn't find any expression with earlier construct also, better look for optimization part, such as codon optimization of gene, IPTG concentrations, induction temperatures and different host vector combinations
Thank you
Agree with Suneelshekar Yapara,
To rule out the possibility of the mutation occurring the promotor/regulatory region of you vector of choice. Sub clone your protein to the original vector (that is before the mutation). This way you do not need to sequence the whole construct and you may be rather sure that the effect comes from the mutation that you have introduced in you cDNA sequence.
If your protein is still not expressed after sub cloning then you go to full optimisation of the expression: IPTG, OD600 at induction, temperature or other strain or you host.
@Stefano
Is that a common problem and needs to be taken into account when "optimizing" protein sequences? Caspase-8 (recognizing the peptide sequence LETD) should only be active in apoptotic cells, at least to my - probably incomplete - knowledge.
Did you check both the soluble and insoluble fractions. It is unlikely, but still possible that your protein became soluble or insoluble due to mutation.
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