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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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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.
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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.

Mentions: Our new expression vector, pCfvtx, Cassette Fused with Venus [3] in the p Trie X1.1-Hygro vector (Novagen), allows for rapid subcloning of basic and fusion cassettes by screening positive colonies using fluorescence (Figure 1d). This vector fixes site 1 and 3 as NcoI and XhoI, respectively, but there are many choices for site 2a and 2b: StuI and SmaI or NheI and SpeI or BamHI and BglII. Since standardization of these specific sites is required to create a basic cassette, they must be added to the domain of interest by PCR and then inserted into the vector. pCfvtx was constructed with a stop codon flanked by two multiple cloning sites (MCS1 and MCS2) upstream of Venus [3], a mutant variant of green fluorescent protein (GFP). When a fragment is subcloned into the vector between MCS1 and MCS2, the stop codon is removed and therefore, a fluorescent cassette is created since it is fused with Venus. As the leak expression of the fusion protein is enhanced by the presence of the T7lac promoter and the absence of the lacI repressor gene [4], positive colonies will be fluorescence on bacterial culture plates (Figure 3a). To create a non-fluorescent cassette, Venus can be removed by cutting with PmeI, performing a self-ligation and then screening for the absence of fluorescence.


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

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

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC183836&req=5

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.
Mentions: Our new expression vector, pCfvtx, Cassette Fused with Venus [3] in the p Trie X1.1-Hygro vector (Novagen), allows for rapid subcloning of basic and fusion cassettes by screening positive colonies using fluorescence (Figure 1d). This vector fixes site 1 and 3 as NcoI and XhoI, respectively, but there are many choices for site 2a and 2b: StuI and SmaI or NheI and SpeI or BamHI and BglII. Since standardization of these specific sites is required to create a basic cassette, they must be added to the domain of interest by PCR and then inserted into the vector. pCfvtx was constructed with a stop codon flanked by two multiple cloning sites (MCS1 and MCS2) upstream of Venus [3], a mutant variant of green fluorescent protein (GFP). When a fragment is subcloned into the vector between MCS1 and MCS2, the stop codon is removed and therefore, a fluorescent cassette is created since it is fused with Venus. As the leak expression of the fusion protein is enhanced by the presence of the T7lac promoter and the absence of the lacI repressor gene [4], positive colonies will be fluorescence on bacterial culture plates (Figure 3a). To create a non-fluorescent cassette, Venus can be removed by cutting with PmeI, performing a self-ligation and then screening for the absence of fluorescence.

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