Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC183836&req=5

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.

Mentions: It should be noted that Venus is the fastest folding and brightest GFP mutant to date [3]. Accordingly, the positive colonies will become fluorescent immediately, whereas other GFP variants may require several days. Second, these fluorescent colonies ensure that the inserted fragment is in-frame and without nonsense mutations. Also, the C-terminal fusion of GFP to target proteins is an effective assay for protein solubility and fold stability – the more fluorescent the fusion protein, the more soluble and well-folded the inserted fragment [5,6]. Lastly, any desired fusion cassette can be designed, such that at each intermediate step, a positive colony is selected by the presence or absence of fluorescence (Figure 2). As only one fluorescent or non-fluorescent colony is needed and the random gain or loss of this property is improbable, fluorescence is a robust reporter that tolerates much of the inefficiency in the subcloning process. In sum, through the use of fluorescence, subcloning is performed rapidly and precisely such that it is possible to efficiently create many fusion cassettes in parallel.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: It should be noted that Venus is the fastest folding and brightest GFP mutant to date [3]. Accordingly, the positive colonies will become fluorescent immediately, whereas other GFP variants may require several days. Second, these fluorescent colonies ensure that the inserted fragment is in-frame and without nonsense mutations. Also, the C-terminal fusion of GFP to target proteins is an effective assay for protein solubility and fold stability – the more fluorescent the fusion protein, the more soluble and well-folded the inserted fragment [5,6]. Lastly, any desired fusion cassette can be designed, such that at each intermediate step, a positive colony is selected by the presence or absence of fluorescence (Figure 2). As only one fluorescent or non-fluorescent colony is needed and the random gain or loss of this property is improbable, fluorescence is a robust reporter that tolerates much of the inefficiency in the subcloning process. In sum, through the use of fluorescence, subcloning is performed rapidly and precisely such that it is possible to efficiently create many fusion cassettes in parallel.

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