how to clone multiple inserts simulataneously - need to make a triple fusion (May/01/2008 )

Hi,
I need to construct a plasmid with three ORFs serially. I want to express three proteins fused together. Is there any way to clone all three ORFs simultaneously into a bacterial vector? I must mention that 2 of the three are more than 1 Kb long.

Do you mean,
all there proteins are expressed as a single fusion protein
Promoter..... mRNA(A-B-C).... protein(ABC)

or

all three proteins are expressed under the same promoter but later become separate proteins
Promoter....mRNA(A-B-C)...... protein A, protein B and protein C

or

three proteins expressed under three different promoters but all contained on the same plasmid


If you are making a fusion one big protein, you will have to build primers which
-contain code for an amino acid loop... to serve as a hinge between proteins.
- you can add a restriction site into the sequence that codes for this loop.
alternatively, the primer can be made to have short stretches of homology, which allow gene fusion by PCR.

If you are looking at the second option, you should look up 2A self cleaving peptide. Place this peptide between the proteins. Once expressed, the self cleaving peptide cuts itself and the large fusion proteins break down to the individual proteins.

The third option would be to cut out/PCR amplify your ORF of proteins including their respective promoters and terminators. Once all three fragment, you can ligate them into your plasmid. In this situation, it is important to identify restriction sites that each fragment contained (and the restriction enzyme that you have) to design the cloning strategy.