strategy to isolate cDNA and express protein - (May/26/2009 )

Hi, I need help. My tutor has given a question which I think I have the right idea but am not 100%

Have ANLN protein, want to examine its location in the cell and what proteins it interacts with. Given all the usual reagents but addtionally a mono-layer of non-small cell lung cancer cells, sense and antisense primers specific for ANLN mRNA expression vector pcDNA 3.1/ His and also some normal lung cells, monoclonal His 6 antibody, anti-mouse HRP antibody and anti mouse FITC. the primer has 5' Bam H1 site on the sense and 5' EcoR1 site incorporating a STOP codon upstream of the RE site.

how do i isolate the cDNA and express the protein in mammalian cells?

I think I need to isolate and produce cDNA then RT-PCR before cloning into the expression vector then need to insert vector into mamalian cell and use tags to identify the protein within the cell?? I am not confident on these processes and need direction!

You are correct in your procedure. Extract the RNA, convert into cDNA and then amplify by PCR (think about types of polymerase you might use and why you might not use Taq). The cut sites in the primers will be amplified and then used to generate "sticky" ends on your PCR product, that can then be ligated into complementary sites in the plasmid after it has also been digested. You will put this into bacteria, select clones, and screen for correct insert sequence. You will then grow up the bacteria and extract the plasmid.

pcDNA3.1 is a mammalian expression vector, meaning that it can express proteins in mammalian cells. You will need to transfect the plasmid containing your cloned cDNA into your cells. You can then look for his-tagged protein (your protein, as opposed to endogenous) using immunocytochemistry and /or western blot - especially if you fractionate your cells.