A fluorescent cassette-based strategy for engineering multiple domain fusion proteins
© Truong et al; licensee BioMed Central Ltd. 2003
Received: 10 April 2003
Accepted: 15 July 2003
Published: 15 July 2003
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.
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.
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.
Conceptually, a fusion protein is constructed by joining two different domains to produce a new chimeric protein, which retains the properties of the individual domains. For instance, a tumor necrosis factor (TNF) inhibitor was created from the fusion of the TNF receptor to the Fc domain of human immunoglobin G as it retains the ability to bind TNF and to be targeted by the immune system . By using the principle of fluorescence resonance energy transfer, protein biosensors can be created from multiple domain fusions with fluorescent proteins to image cellular events such as Ca2+ signaling, phosphorylation, and caspase proteolytic cleavage . In practice, such fusion proteins are created by inserting PCR products of the individual domains into an expression vector at the available restriction endonuclease sites. Previously, the flexibility of design is compromised as the choice of insertion sites limits the possible locations for future fusions into the same expression vector. In turn, many initially unplanned but simple extensions to existing fusion proteins cannot be constructed because available sites are exhausted or incompatible. As this issue is exacerbated when constructing multiple domain fusion proteins, we have created a new expression vector for subcloning using a cassette-based strategy. A basic cassette contains the sequence of an individual domain that can be recombined with other cassettes irrespective of order, without the progressively more complex management of sites. Therefore, the creation of a cassette library of commonly used domains facilitates the rapid prototyping of multiple domain fusion proteins that can perform numerous functions.
Results and discussion
Standard vector structure of the cassette
pCfvtx embodies the standard vector structure and allows fluorescence screening
Fluorescence screening with Venus
It should be noted that Venus is the fastest folding and brightest GFP mutant to date . 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.
Protein purification cassettes
To demonstrate the utility of our cassette-based strategy, we first applied it to protein expression/purification systems, which often involve either an N-terminal or C-terminal fusion of the target protein with an affinity tag. Cassettes were made using two popular tags – 6xHis (Qiagen) and Glutathione S-transferase (GST) tag (Pharmacia) . The N-terminal fusion of the 6xHis tag to Venus allows binding to Ni-NTA (nickel-nitrilotriacetic acid) agarose beads, however, a simple elution yields an impure sample (Figure 3b). The additional C-terminal fusion of GST to Venus allows binding of the previous elution to GST sepharose beads. Since the affinity tags flank the target protein and it is unlikely that a protein will non-specifically bind to both affinity beads, only full-length fusion proteins will be eluted from the GST beads. Note that the newly created 6xHis-Venus-GST fusion cassette is itself a useful affinity tag that additionally could be used to estimate protein expression greater than ~1 nM as fluorescence intensity from the Venus domain is linearly proportional to target protein concentration. Finally, the flexibility of our cassette-based strategy opens new opportunities for the design of tandem affinity purification (TAP) tags , which were useful in protein complex purification in the yeast proteome . The customization of TAP tags is desirable as the same affinity tag may not be suitable for all organisms .
Protein subcellular targeting cassettes
The creation of protein biosensors has allowed the observation of signaling events in single cells [11–13]. Such events are often isolated to subcellular organelles such as the nucleus or endoplasmic reticulum and therefore, the ability to easily localize biosensors to these sites is important. The localization of proteins to specific organelles relies on vital cellular mechanisms that recognize leader sequences and signal peptides . If a protein (such as the 6xHis-Venus-GST protein) is expressed in the cell without any localization peptides, it will be found inside the cytoplasm (Figure 3c). To localize a target protein to the nucleolus, a cassette was created containing the protein transduction domain of human immunodeficiency virus (HIV) Tat . When this cassette was N-terminally fused to Venus and transfected into COS-7 cells, fluorescence was most intense in the nucleolus (Figure 3d). To localize to the lumen of the endoplasmic reticulum, a cassette was created containing the leader sequence from interleukin-4 and another cassette was created with the KDEL retention signal. When these cassettes were fused N- and C-terminally to Venus, it localized to the endoplasmic reticulum (Figure 3e). In summary, the creation of these cassettes allows the flexibility of localizing any cassette in our library to those organelles.
List of cassettes
Fundamental vector for rapidly inserting PCR products using a fluorescence assay to create basic cassettes
Venus for acquisition of fluorescence
6xHis affinity tag
GST affinity tag
6xHis affinity tag that also allows protein expression estimation by fluorescence
A double affinity tag using 6xHis and GST for improved purity and protein expression estimation by fluorescence
Subcellular organelle targeting
HIV TAT protein tranduction domain for targeting to the nucleolus or peptide-mediated delivery of proteins to the nucleus
Interleukin-4 signal peptide for secretion of target protein (alone) or for localization to the ER (with KDEL retention)
KDEL retention signal for localization the ER (with signal peptide)
A fluorescent marker for the nucleolus
A fluorescent marker for the ER
Vectors were transformed into E. coli strain DH5α and plated on LB (Luria Broth) agarose with 100 μg/mL ampicilin. The culture plates were then incubated overnight at 37°C. Venus fluorescence was observed on the culture plate using the Lighttools Illuminatool Tunable Lighting System equipped with a 535 nm viewing filter and 488 nm/10 nm filter cup.
Construction of the pCfvtx vector
To create the pInsvtx intermediate vector, Venus was PCR amplified from the pVenus vector  using primers Insv-sense (5'-CATGCCATGGGCCTGACTAGTAGGCCTGCTAGCCTGTTTAAACTGGTGAGCAAGGGCGAGGAGCTG-3') and Insv-antisense (5'-CCGCTCGAGTTACAGTTTAAACAGGGCGGCGGTCACGAACTCCA-3'). The Insv-sense contained NcoI, SpeI, NheI and PmeI sites, while Insv-antisense contained PmeI and XhoI sites. The fragment was subcloned into the pTriEx1.1-Hygro vector (Novagen) at the NcoI and XhoI sites by selecting a fluorescent colony. The pInstx intermediate vector was created by self-ligating after cutting at the PmeI site of pInsvtx and a non-fluorescent colony was selected. Finally, to create the pCfvtx vector, a fragment containing the multiple cloning sites (SpeI, BamHI, StuI, BglII, SmaI, NheI) sandwiching a stop codon was created using 5'-end phosphorylated primers Mcs-sense (5'-CTAGTGGATCCAGGCCTTAAAGATCTCCCGGGG-3') and Mcs-anti-sense (5'-CTAGCCCCGGGAGATCTTTAAGGCCTGGATCCA-3'). Mcs-sense and Mcs-antisense were self-hybridized and subcloned into the pInsvtx vector at the SpeI and NheI sites. A non-fluorescent colony was selected.
Construction of the subcellular targeting vectors
To create the pVentx vector, Venus was amplified using primers Ven-sense (5'-CATGCCATGGGCCTGACTAGTGTGAGCAAGGGCGAGGAGCTG-3') and Ven-antisense (5'-CCGCTCGAGTTAGCCGCTAGCGGCGGCGGTCACGAACTCCA-3'). The Ven-sense contained NcoI and SpeI, sites, while Ven-antisense contained XhoI and NheI sites. The fragment was subcloned into the pInstx vector at the NcoI and XhoI sites by selecting a fluorescent colony. To create pTatvtx, 5'-end phosphorylated primers Tat-sense (5'-CATGGGCCTGACTAGTTACGGCAGGAAGAAGAGGAGGCAGAGGAGGAGGGGGG-3') and Tat-anti-sense (5'-CTAGCCCCCCTCCTCCTCTGCCTCCTCTTCTTCCTGCCGTAACTAGTCAGGCC-3') were self-hybridized and subcloned into the pCfvtx vector at the NcoI and NheI sites. Similarly, to create the pIl4vtx vector, 5'-end phosphorylated primers il4-sense (5'-CATGGGCCTGACTAGTCAGCTGCTGCCGCCCCTGTTCTTCCTGCTGGCCTGCG-3') and il4-anti-sense (5'-CTAGCGCAGGCCAGCAGGAAGAACAGGGGCGGCAGCAGCTGACTAGTCAGGCC-3') were self-hybridized and subcloned into the pCfvtx vector at the NcoI and NheI sites. The pIl4tx was created by self-ligating after cutting at the PmeI site of pIl4vtx. To create the pVkdeltx intermediate vector, Venus was amplified using primers Ven-sense and kdel-antisense (5'-CCGCTCGAGTTACAGCTCGTCCTTACTAGTGGCGGCGGTCACGAACTCCA-3'). The kdel-antisense contained XhoI and SpeI sites. The fragment was subcloned into the pInstx vector at the SpeI and XhoI sites. The pKdeltx was subcloned by cutting and self-ligating at the SpeI site. To create pVenkdeltx vector, pVentx was cut with NcoI and NheI and the fragment was subcloned into pKdeltx at NcoI and SpeI sites. To create pIl4venkdeltx, pVenkdeltx was cut with SpeI and XhoI and the fragment was subcloned into pIl4tx at the NheI and XhoI sites.
Construction of the 6xHis-Venus-GST cassette (pHisvengsttx vector)
To create pGstvtx, GST was amplified from pGEX2T (Invitrogen) using primers gst-sense (5'-GACTAGTATGTCCCCTATACTAGGTTATTG-3') and gst-antisense (5'-GAAGATCTATCCGATTTTGGAGGATGGTCG-3'). The gst-sense and gst-antisense contained SpeI and BglII sites, respectively. The fragment was subcloned into the pCfvtx vector at the SpeI and BglII sites. pGsttx was created by cutting and self-ligation at the PmeI site. To create the pHisvtx vector, 5'-end phosphorylated primers his-sense (5'-CATGGGCCTGACTAGTGGCAGCAGCCACCACCACCACCACCACAGCAGCGGCG-3') and his-anti-sense (5'-CTAGCGCCGCTGCTGTGGTGGTGGTGGTGGTGGCTGCTGCCACTAGTCAGGCC-3') were self-hybridized and subcloned into the pCfvtx vector at the NcoI and NheI sites. pHistx was created by cutting and self-ligation at the PmeI site. To create pHisventx vector, pVentx was cut with SpeI and XhoI and the fragment was subcloned into the pHistx at NheI and XhoI sites. To create pHisvengsttx, pHisventx was cut with NcoI and NheI and the fragment was subcloned into pGsttx at the NcoI and SpeI sites.
Transfection and imaging of tissue cultures
COS-7 cells were transfected with GeneJuice (Novagen). Between 2 and 5 days after transfection, cells were imaged at 22°C on an Olympus IX70 microscope with a CCD camera (MicroMax 1300YHS) controlled by MetaMorph 4.5r2 software (Universal Imaging).
The vector pVenus was generously provided by A. Miyawaki. We thank K. Tong for technical assistance. This work was supported by grants to KT from the Canadian Institutes of Health Research (CIHR) and to MI from the Cancer Research Society, Inc. and the Institute for Cancer Research of the CIHR.
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