Is it appropriate to place the ATG codon in front of the gene of interest, since there is a secretion signal that has its own ATG in front of this gene? I need my protein of interest to be secreted into the medium, so I used a vector with an alpha factor. If I clone the gene of interest as a gene for alpha factor with ATG, is it possible that Pichia pastoris will recognize 2 reading frames and the protein of interest can be produced intracellularly? Or is it better to clone the gene of interest without its own ATG so that I can be sure that the yeast will be read an alpha factor and the gene of interest as one reading frame and the protein will be secreted into the medium? Thanks in advance for the answers
In eukaryotes primarily the first start codon will be used, otherwise every internal Met would serve to initiate a protein. So it probably doesn't matter whether or not you have an ATG at the start of your ORF when cloned in frame with the signal sequence.
However it sounds like the protein you intend to make is normally not secreted, there are many examples where non-secreted (cytoplasmic) proteins can not be secreted even if you add a signal sequence. So just be aware that this may or may not work for you.
As Michael said, the post-signal methionine will be mostly cosmetic. The distance between the initial methionine and the TATA box, which is required for translation initiation, has a rather narrow sweet spot, hence it's highly unlikely (in statistical terms) that translation is initiated at the second methionin located behind the signal peptide and even in these rare cases you won't care as you'll harvest only the secreted protein.
As long as it's a soluble protein that doesn't extensively multimerize, you should be fine. Trial and error.
Michael J. Benedik Thank you for your response! It is protein which is secreted in its original organism. That is why I need P. pastoris to secrete to medium and glycosylate it
Andre Marschall Thank you for your response Andre! I will try to express it with ATG and also without ATG :)
Bohuš Kubala Generally adding a signal sequence to the front of a protein that already has a signal sequence may not be a good idea. And since your question is about the ATG start codon this suggests you may be including the signal sequence. I would either try expressing the protein using only its native signal (They often but not always work cross species) OR remove the signal and attach the mature reading frame to the vector in-frame with the vector signal sequence.
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