I purchased the human clone of this gene and sub cloned it into a Xenopus oocyte plasmid (it contains Xenopus oocyte beta globin to enhance the expression in Xenopus oocyte) with a polyA site. But no functional activity of this protein was detected (I repeated the experiment 3 times). I sequenced the clone and it is correct.
From the literature, I know that its rodent and fish homologues, and some of its family members in human were successfully expressed in Xenopus oocyte with detectable activities without expressing their known co-factor(s). I found an unpublished dissertation work online saying that they couldn't detect function of this human clone in Xenopus oocyte either using a different functional assay.
I ordered antibody of this protein hoping to see whether it didn't express or its function was inhibited for some reason. While waiting for the antibody (it will take weeks), is there anything that I can do to help figure out the reason why I didn't detect its function?
I'd assume that the codon usage is ok within vertebrates, but better double check (-> codon usage database). A few infrequent codons are enough to mess up everything. Then check if everything required for expression is really there: promoter, translation initiation sequence (Kozak consensus element, start and stop codon, reading frame, polyA-signal).
Can you detect the mRNA by qPCR?
Have you tried to express an unrelated protein like e.g. GFP the same way?
@Wolfgang Hello, thanks for answering. I forgot to mention that I injected cRNA made by IVT into the oocytes. So I guess it won’t be necessary for qPCR. I’ve had many other proteins including GFP expressed in oocytes using the same vector. The plasmid has a promoter, a polyA site,kozak sequence. The ORF is correct based on the transcript on NCBI.
Can you inject a mix with another cRNA that is known to work and check the expression?
Hm. What about cryptic splice sites? Not sure how to detect that, maybe trying to rescue the sequence by PCR and sequence the shorter bands, if any.
Or some unexpected signal sequences.
But all that is just wild guessing.
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