A fluorescent cassette-based strategy for engineering multiple domain fusion proteins.
BACKGROUND
The engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all.


RESULTS
Therefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence.


CONCLUSIONS
Finally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.
Truong
Kevin
K
Department of Medical Biophysics, University of Toronto, Toronto, M3N 1L6, Canada. ktruong@uhnres.utoronto.ca
Khorchid
Ahmad
A
Ikura
Mitsuhiko
M
D003001:Cloning, Molecular / Q000379:methods; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D049452:Green Fluorescent Proteins; D006639:Histidine / Q000235:genetics; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine; D005982:Glutathione Transferase