Cloning genetic sequences of proteins. - (Jun/21/2012 )
Hi guys, Im an undergrad student and i am having some trouble working out the basis for my upcoming project.
I have been told I have a protein that is purified to homogeneity and that i need to work out how to clone the genetic sequence for the protein.
The information given is as follows:
it is a single polypeptide protein, that is soluble.
has been isolated from cultured human fibroblast cells (skin cells) that are maintained in tissue culture.
Expression of the protein can be induced in the fibroblast cultures by briefly exposing the cells to 40oC for 30 minutes and waiting 2 hours.
The homologous protein from pigs has been purified and characterised. The pig protein appears to carry out the same function as the human protein and a cDNA and antibodies raised against the pig protein are available for you to use.
I know i need a plasmid but I am not sure what type, not used so much to dealing with mammalian proteins. Do i need one with particular selectable markers or a certain ori?
Thanks in advance if anyone can lend me a hand
the thing is that it is very likely that the human plypeptide is already cloned; so, a genetic data bank alignment with the pig cDNA will probably give the required information about the human cDNA;
you can subclone it into a mammalian expression vector optionally with a reporter gene or tag and/or a selection marker to select positive cells...