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

Truong K, Khorchid A, Ikura M - BMC Biotechnol. (2003)

Bottom Line: Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biophysics, University of Toronto, Toronto, M3N 1L6, Canada. ktruong@uhnres.utoronto.ca

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.

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Related in: MedlinePlus

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.
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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.

Mentions: Our cassettes must have a standard vector structure where a domain(s) is flanked by cut sites 1 and 2a at the 5' end and 2b and 3 at the 3' end (Figure 1a). Site 1 and 3 can be selected arbitrarily, but site 2a and 2b must be derived from different restriction enzymes producing blunt or compatible cohesive ends. For example, there are two ways to create the AB fusion cassette from cassette A and B (likewise for the BA fusion cassette): ligate the insert from cassette B (site 2a and 3) to the host cassette A (site 2b and 3) (Figure 1b) or ligate the insert from cassette A (site 1 and 2b) to the host cassette B (cut site 1 and 2a) (Figure 1c). Since the ligation point of site 2a and 2b produces the recognition site of neither, it cannot be cut with either restriction enzymes. Therefore, the AB fusion cassette has the same standard vector structure and can be used for further fusion following the same concept. Note that if no more than one of the four enzyme sites are found inside the domain sequence of the cassette, it is still possible to create any fusion because cassettes can be fused on either the 5' or 3' end.


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

Truong K, Khorchid A, Ikura M - BMC Biotechnol. (2003)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183836&req=5

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.
Mentions: Our cassettes must have a standard vector structure where a domain(s) is flanked by cut sites 1 and 2a at the 5' end and 2b and 3 at the 3' end (Figure 1a). Site 1 and 3 can be selected arbitrarily, but site 2a and 2b must be derived from different restriction enzymes producing blunt or compatible cohesive ends. For example, there are two ways to create the AB fusion cassette from cassette A and B (likewise for the BA fusion cassette): ligate the insert from cassette B (site 2a and 3) to the host cassette A (site 2b and 3) (Figure 1b) or ligate the insert from cassette A (site 1 and 2b) to the host cassette B (cut site 1 and 2a) (Figure 1c). Since the ligation point of site 2a and 2b produces the recognition site of neither, it cannot be cut with either restriction enzymes. Therefore, the AB fusion cassette has the same standard vector structure and can be used for further fusion following the same concept. Note that if no more than one of the four enzyme sites are found inside the domain sequence of the cassette, it is still possible to create any fusion because cassettes can be fused on either the 5' or 3' end.

Bottom Line: Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Biophysics, University of Toronto, Toronto, M3N 1L6, Canada. ktruong@uhnres.utoronto.ca

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.

Show MeSH
Related in: MedlinePlus