Standard vector structure of the cassette
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.
pCfvtx embodies the standard vector structure and allows fluorescence screening
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.
Fluorescence screening with Venus
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.
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) [7]. 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 [8], which were useful in protein complex purification in the yeast proteome [9]. The customization of TAP tags is desirable as the same affinity tag may not be suitable for all organisms [10].
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 [14]. 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 [15]. 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.