A fluorescent cassette-based strategy for engineering multiple domain fusion proteins

Abstract

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.

Figure 1

The cloning methodology. (A) Design of cassette A and B. Creation of the AB fusion cassette by (A) a C-terminal fusion to cassette A and (B) a N-terminal fusion to cassette B. (D) Schematic diagram of the pCfvtx vector. pTriEx1.1-Hygro (Novagen) was chosen as the base vector because it allows expression in both prokaryotic and eukaryotic cells.

Figure 2

Flow diagram of our fluorescent cassette-based strategy to construct the AB fusion cassette. First, the fluorescent cassettes A and B are created by insertion of the respective domains into the pCfvtx vector. Then, cassette A and B is created by the excision of Venus by PmeI restriction and then self-ligation. The path of the thick arrows highlight the fusion steps required for creating the AB fusion cassette. The fluorescent cassette B is ligated to cassette A to create a fluorescent cassette AB. The final AB fusion cassette is created by the excision of Venus as described previously.

Figure 3

Fluorescence screening, protein purification and subcellular localization. (A) Fluorescence screening in bacterial colonies using the leak expression of the target protein fused with Venus. A non-fluorescent colony is identified by arrow 1; a fluorescent colony, by arrow 2. (B) The purification of the 6xHis-Venus-GST construct (identified by the arrow). Lane MW is the molecular weight marker; lane 1, the cell lysate; lane 2, the elusion from the Ni-NTA column; lane 3, the elusion from the GST column. All vectors were transfected into COS-7 cells for fluorescence imaging. The 10× magnification of (C) the cytoplasmic distribution of the 6xHis-Venus-GST and (E) the endoplasmic distribution of Venus N-terminally fused with the interleukin-4 leader sequence and C-terminally fused with the KDEL retention signal. (D) The 40× magnification of the nucleolar distribution of Venus N-terminally fused with the HIV Tat domain.