Dear all,
I want to express pectin lyase D gene 1122bp (A. niger) in pichia pastoris X-33 using PpicZalpha A vector.
In silico analysis showing translation of whole sequence in to amino acids, this is similar to reported protein in NCBI.
But further analysis of 1122 bp sequence I found 5’ splice with score > 80 at 396th position in my pectin lyase sequence.
Please tell me whether it’s really going to splice in Pichia or not?
If yes, then my protein will not express by transformed Pichia pastoris.
And how to overcome this problem?
I do not know how this protein will express in Pichia, but you can probably overcome any splicing problems by modifying the gene. I would try site-directed mutagenesis to eliminate the splice site by changing the nucleotide sequence. Make sure that you retain the correct amino acid coding by using synonymous codons where you make changes.
Hi Ymtiyaz,
Have you found out whether or not 'splice' occurred?
One way you can find out is design some testing primers and do RT-PCR to see if you can get full-length product, or part of the mRNA got spliced away. By using the results from this way and Nicholas' suggestion, you might able to write a mini-paper.
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