I introduced a point mutation in my protein and cloned the mutated gene into the pCold vector. After sequencing, I observed a gap of 2 base pairs upstream of the mutation site. Considering the gap in the sequence, I translated the nucleotide sequence to assess whether a frameshift had occurred. The translation revealed a premature stop codon, indicating a truncated protein. According to the Expasy ProtParam analysis, the expected molecular weight of the truncated protein was approximately 56 kDa. However, upon induction and purification, the expressed protein appeared to be of a similar size as the native (non-mutated) protein on SDS-PAGE. So, I should consider this as sequencing error of I need to do further experiments for validation?
It would be easier to assess if there has been a sequencing error if you post the original .abi sequence file
That sequence looks really good, I don't think there was an error in the Sanger. I think your bacteria has a DNA deletion. Note: a 2 bp deletion will *always* cause a frameshift. Indels that are multiples of 3 keep the mRNA in frame (with one or more amino acids added or removed).
I'm not a protein expert, but I would not trust the estimated protein sizes from that gel. Especially not if you have an "estimated" protein size.
Do you have other bacterial colonies to test out? Sequence the DNA first before you start in on protein expression.
Words of wisdom for plasmids: "Never trust that the sequence is correct, always verify".
Good luck!
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