The Return of the Native

Some of the most widely used ORF resource clones dispense with native termination codons. This makes perfect sense if the goal is to permit rapid exchange between various expression vectors, since the initiation and termination codons can be provided by the plasmid backbone in a wide variety of contexts—including N-terminal and/or C-terminal fusions. However, this flexibility comes at the cost of being unable to express the protein in its true native form. This limitation can be a showstopper; for instance, C-terminal ends of receptor tyrosine kinases cannot have extraneous nonnative residues if accurate signaling analysis is to be performed. Itoh et al. describe now how to have the best of both worlds. In their ORFeome vector strategy, the native termination codon is present but easily removable. The trick involves the use of a type-IIS restriction enzyme: thanks to strategic placement of the recognition site, the termination codon can be excised by a digestion/self-liga-tion procedure. Because the strategy does not rely upon PCR, expensive and time-consuming resequencing is minimized. Importantly, users of this modified entry clone sacrifice nothing in convenience, since the vector provides the choice of standard recombinational cloning or homing endonuclease-mediated recombination for vector exchange reactions. -Page 44

Vector exchange systems promise to revolutionize molecular biology due to their capacity for high-throughput parallel transfer of cloned complementary DNA (cDNA) into multiple functional vectors. These systems use specific recombination reactions; examples include manipulations based on phage λ integration/excision at att sites (1) (commercialized as the Gateway® system, www.invitrogen.com/content.cfm?pageid=4072), the Cre-LoxP system (www.clontech.com/clontech/products/families/creator/index.shtml), and recombination mediated via homing endonuclease (HE) sites (2). Notably, the Gateway system has been used for ORFeome projects to produce collections of entry clones that can be transferred into destination vectors suitable for in vitro expression, two-hybrid analysis, and mammalian expression (3). The Gateway ORFeomes allow straightforward expression of native proteins as well as N-terminal and/or C-terminal fusion proteins, because the initiation and termination codons for each cloned open reading frame (ORF) are provided by the destination vectors. However, as noted by Brasch et al., in some cases it is necessary that the C-terminal end of the protein be in its native form, such as in the expression of receptor tyrosine kinases for signaling analysis (4).

For that reason, we decided to generate ORF-resource clones that contain the protein's native termination codon. Each clone is flanked by HE sites and is placed in a Gateway entry vector. The HE sites function as extremely rare cutter sites that are suitable for cloning and/or excision of the fragment. Previously, Asselbergs et al. applied HEs for cloning, since these enzymes are restricted to uncommon genome sequences and do not, therefore, cause random cleavage (5). We have also used HE-cleaving sequences as cloning sites to excise the appropriate DNA fragments for the large-scale construction of expression plasmids of Pyrococcus horikoshii OT3 ORFs and found that this strategy resulted in a high success rate (Reference 6) and unpublished data). In addition, the sites allow maximum flexibility in destination vector choice, permitting vector transfer by HE recombination or att site-mediated recombination.

Although our combined Gateway-HE entry vector offers significant flexibility for a variety of downstream applications, the presence of the native termination codon in each ORF means that the system cannot be directly used for the production of proteins with C-terminal fusions. Currently, existing protocols eliminate the termination codon by PCR or by some other mutation protocol before the vector exchange step. However, even when using high-fidelity PCR, there are unwelcome reading frame mutations, that require multiple resequencing of clones before protein expression. Here, we describe a method that does not involve PCR to eliminate the termination codon and shows the strategy's suitability for generating C-terminal fusion protein expression clones.

We developed our method for a project aimed at generating cellular localization data by fusing green fluorescent protein (GFP) to the C terminus of selected ORFs. Here, we show the case of human p53 gene, although similar results were obtained from other ORFs (data not shown). First the ORF in question must be cloned into the Gateway-HE entry vector. As shown in Figure 1, p53 ORF was amplified by high-fidelity PCR with Platinum® Pfx DNA polymerase (Invitrogen, Carlsbad, CA, USA) with 25 cycles of the following thermal program: 95°C for 1 min, 60°C for 1 min, and 68°C for 4 min. The specific primers used were 5′-gaaggagccgccaccATGACTGCCA TGGAGGAGT-3′ and 5′-caattgttcacag gaaacTCAGTCTGAGTCAGGCCC-3′, in which uppercase letters denote the bases specific for p53. These primers are based on our report (7) with some modifications. A second PCR was done with adaptor primers carrying HE sites: forward primer 5′-CACCGCTAGGGATAACAGGGTAATAgaaggagccgcc accatg-3′ [bases in bold are essential for directional cloning with pENTR-TOPO® cloning kit (Invitrogen); underlined are bases forming the site for the HE I-SceI; lowercase letters are bases that are identical to those in the forward primer used in the first PCR] and reverse primer 5′-TGCTACCTTA AGAGAGGAGcaattgttca caggaaactca-3′ (underlined bases represent the bottom strand of the recognition site for the HE I-PpoI; lowercase letters are bases that are identical to those in the reverse primer used in the first PCR; bold letters indicate sequences making up the bottom strand of the recognition site for restriction enzyme PpiI). The PCR conditions were identical to the first amplification, and the final PCR product was purified with Geneclean® III kit (Qbiogene, Irvine, CA, USA) and cloned by the pENTR-TOPO cloning kit according to the manufacturer's manual; the resulting Gateway-HE entry clone was confirmed by sequencing.

Figure 1. Construction for Gateway-HE entry clone. (Click to enlarge)

As shown in Figure 2, the plasmid was then subjected to the termination codon elimination step by digestion at the PpiI recognition site, which was introduced by the reverse primer during the second PCR amplification. PpiI is a type-IIS restriction enzyme (Fermentas, Burl-ington, ON, Canada) with a recognition site [(7/12) GAACNNNNNCTC (13/8)]. This recognition site is found downstream of the termination codon, as shown in Figure 1 and Figure 2. This enzyme was selected because the recognition sequence is longer than other type-IIS enzymes, the cutting position is fixed, and there are no such sites in pENTR. In this case, one of the cutting positions is the termination codon position. After digestion, a portion of the reaction (about 0.5 µg) was subjected to blunting by T4 DNA polymerase (Takara Bio, Shiga Japan) with 0.2 mM dNTPs and the manufacturer's buffer, followed by self-ligation at a dilute concentration (approximately 1 ng/µL) with T4 DNA ligase (Takara Bio) with 1 mM ATP at 25°C for 1 h. The ligation solution was then used for Escherichia coli transformation.

Figure 2. Termination codon elimination using a type-IIS restriction enzyme. (Click to enlarge)

The plasmid DNA was then used for a LR reaction of the Gateway system with destination vector pcDNA-DEST47 (Invitrogen) to generate a C-terminal GFP fusion protein (following manufacturer's recommendations). After the transformation, plasmid DNA was extracted from the resulting colonies and used for transfection into NIH 3T3 fibroblast cells. Analysis by fluorescence microscopy (Figure 3) showed that the expressed p53-GFP fusion protein exhibited the expected localization pattern of normal p53 protein (8).

Figure 3. p53-GFP localization. (Click to enlarge)

As shown here, we have developed a non-PCR-mediated termination codon elimination method with a type-IIS restriction enzyme. In cases where a PpiI site is located in the ORF, we can use similar restriction enzymes, such as PsrI (SibEnzyme, Novosibirsk, Russia). Thus, starting from an entry clone that contains sites for both att and HE-mediated recombination, and that expresses the protein with its native C terminus, we can use this method to generate quickly a clone that is compatible with transfer to a destination vector that would add a C-terminal fusion protein. In the traditional approach, each expression clone (native C terminus and C-terminal fusion protein) must be PCR-amplified, which requires resequencing to verify the absence of mutations. For large-scale analyses, this would be an expensive process. By contrast, if the described strategy is used, only one entry clone needs to be resequenced, so that only half the number of sequencing reactions would be required to evaluate each entry clone.

To avoid the use of topoisom-erase-based cloning, the step in which the ORF-containing PCR product is subcloned into pENTR could potentially be replaced by a third PCR amplification with adapter primers that add att sites. The resulting PCR product could then be placed in a Gateway vector by recombinational cloning. Although we have not tested this alternative procedure, a proposed workflow is presented in Supplementary Figure S3 (supplementary material is available online at www.BioTechniques.com).

Finally, even if not for the large-scale project, this strategy would be a good choice for the construction of vector-exchange entry clones. Using this entry clone, native, N-terminal, and C-terminal fusion protein expression plasmids can be achieved without PCR. Also, every cloned ORF fragment can be cut out by HE digestion without PCR. Thus, this technique would be helpful to construct universal entry clones that can be used for various types of protein expression purposes. Especially for the large-scale preparation, like ORFeome analysis projects, this strategy would be expected to significantly reduce the cost and time involved in resource preparation.