Cloning incomplete genes - (Apr/16/2007 )
I recently heard about a cloning technique, I am a bit sceptical about and wanted to know if other people also do that:
When cloning a gene into an expression-vector an internal restriction site of the desired gene is used, so a few bases at the 5`end of the coding sequence are lost (start codon is on the vector, gene is in frame). Allegedly the missing bases / aminoacids do not matter and it might even be helpful because the start of the protein is often hydrophobe and may hinder solubility of the latter expressed protein.
Do you agree with that / also routinely do that ?
(I would still worry about losing some functionality, but maybe I am wrong.)
Please let me know about your experiences !
Most likely this works in the majority of cases. It seems a bit sloppy to me, however. You can use COG to locate the conserved motifs in your protein, and verify that the motif, at least, is complete. It seems as if the the technique would rarely work well, given the requirement for losing only a few bases and the need for cloning in-frame.
I have never used this to clone a gene into expression vector. IMHO, this will take even more requirement (close, appropriate internal RE site, in-frame...) than just doing a proper cloning. Also, I think it depends on the purpose of the cloning. E.g., if you only want to express protein to purify as antigen for making antibody, it usually does not matter much if you lose a few a.a. But if you want to express proteins in cell for morphological or functional study, it could make big differences, especially if the first few a.a are necessary for the protein to, say, localize correctly, e.g Arf proteins...