Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products

In this study, we established new systematic protocols for the preparation of cDNA clones, conventionally termed open reading frame (ORF) clones, suitable for characterization of their gene products by adopting a restriction-enzyme-assisted cloning method using the Flexi® cloning system. The system has following advantages: (1) preparation of ORF clones and their transfer into other vectors can be achieved efficiently and at lower cost; (2) the system provides a seamless connection to the versatile HaloTag® labeling system, in which a single fusion tag can be used for various proteomic analyses; and (3) the resultant ORF clones show higher expression levels both in vitro and in vivo. With this system, we prepared ORF clones encoding 1929 human genes and characterized the HaloTag-fusion proteins of its subset that are expressed in vitro or in mammalian cells. Results thus obtained have demonstrated that our Flexi® ORF clones are efficient for the production of HaloTag-fusion proteins that can provide a new versatile set for a variety of functional analyses of human genes.


Introduction
The entire human genome sequence has allowed us to create a provisional catalog of human transcripts and proteins by a combination of computational predictions and information from experimentally collected cDNA sequences. 1 Although approximately 23 000 protein-coding genes have been assigned to the human genome by the Ensembl database (NCBI built 36, Ensembl 48; http://www.ensembl.org/ Homo_sapiens/index.html), 2 the functional roles of the respective gene products remains to be experimentally explored. Because of the importance of these studies, many groups have started to collect a set of protein-coding sequences, conventionally termed open reading frames (ORFs), in a genomewide manner, so-called 'ORFeome cloning'. ORF clones serve as versatile reagents for the functional and structural studies of proteins. 3 -5 Therefore, various systems have been developed to enable efficient large-scale cloning and expression of ORF clones. Currently available systems rely on either site-specific recombinases or rare-cutting restriction enzymes for the transfer of ORFs between expression vectors.
These systems allow the correct orientation and reading frame to be maintained after transfer. 6 -8 The Gateway and Creator cloning if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org systems, which are representative of the recombinasebased systems, transfer ORFs by l-att recombination using the 25-bp attB site, and Cre-loxP recombination using the 34-bp lox-P site upstream of the ORF, respectively. 7 On the other hand, restrictionenzyme-based systems are less frequently used at present, although a commercial system has recently emerged that is as efficient as those based on recombinases. The Flexi w cloning system can manipulate ORFs using rare-cutting restriction enzymes, SgfI and PmeI, after the addition of these 8-bp restriction sites to the flanking 5 0 and 3 0 ends of the ORFs, respectively. 8 We previously prepared a set of ORF clones for human KIAA genes using a Gateway-type vector. 9 Furthermore, we created the ORF clones as fusion proteins with green-fluorescent protein to analyze the subcellular localization of the proteins in cultured cells and to evaluate the in vitro expression of ORF clones. 9 However, when we wish to carry out alternative functional studies beyond bioimaging, the Gateway system requires re-construction of appropriate expression plasmids for the creation of differently tagged proteins. This creates a serious bottleneck in the use of our large ORF clone set for functional genomics analysis. Thus, we started to look for a more versatile tag system that would allow us to carry out both bioimaging and biochemical experiments using a single tag. Within this context, we considered a recently emerging technology, commercially designated as HaloTag w technology. 10 This technology is quite attractive since HaloTag can be used not only for bioimaging, but also for various proteomic applications involving fusion protein immobilization. Although similar technologies have been developed, such as SNAP-tag which uses a modified human O 6 -alkylguanine DNA alkyl transferase as the tag, 11,12 we preferred HaloTag w technology because we felt it may be more suitable for studies in mammals. Haloalkane dehalogenase, the enzyme from which HaloTag is derived, does not exist in mammalian cells, unlike O 6 -alkylguanine DNA alkyl transferase. Since HaloTag w technology was available in the Flexi w cloning system, we were strongly motivated to examine whether the Flexi w cloning method was suitable for preparation of a large number of ORF clones, and whether it would be useful for downstream functional characterization.
In this study, we prepared nearly 2000 ORF clones using the Flexi w cloning method, and the resultant ORF clones were examined with respect to production in cell-free protein synthesis systems as HaloTagfusion proteins. The results indicated that the Flexi w cloning method was as efficient as the Gateway cloning method and that the Flexi ORF clones consistently produced equal or larger amounts of HaloTag-fusion proteins than Gateway clones in in vitro protein production systems. We also examined the subcellular localizations of 40 HaloTag-fusion proteins in HEK293 cells and confirmed that the results were equivalent to those of the corresponding Monster Green w Fluorescent Protein (MGFP)-fusion proteins. Taken together, these results indicate that the Flexi ORF clone set can perform efficient functional analyses of human genes and provide an alternative resource for the human ORFeome project.

Preparation of Flexi ORF clones
The ORF of interest was amplified from 1 to 4 ng of a plasmid DNA using 0.5 units of PrimeSTAR TM HS DNA polymerase (Takara Bio, Shiga, Japan) in a 20-mL PCR reaction mixture that included 0.2 mM each of four dNTPs and 0.5 mM each of the genespecific primers (5 0 -CCCCGCGATCGCCATG N 17 -3 0 and 5 0 -CCCCGTTTAAAC N 20 -3 0 , where N 17 indicates a 17 nt-sense sequence downstream from the start methionine codon of ORF and N 20 indicates a 20 nt-anti-sense sequence upstream from the stop codon), under the following PCR conditions: 958C for 2 min/30 cycles of 988C for 10 s and 688C for 3 min/688C for 5 min. In some cases, 0.5 units of KOD-Plus-DNA Polymerase (Toyobo, Osaka, Japan) was used in a 25-mL PCR reaction mixture that included 0.2 mM each of four dNTPs, 1 mM MgCl 2 and 0.3 mM each of the gene-specific primers under the following PCR conditions: 948C for 2 min/30 cycles of 988C for 10 s, 558C for 30 s/688C for 3 min. One-third of the resultant amplified PCR products was digested by SgfI and PmeI in a 10-mL reaction mixture of Flexi w buffer with 1Â Flexi w Enzyme Blend (SgfI and PmeI) (Promega) for 30 min at 378C after purification of the PCR products using Wizard SV 96 PCR Clean-Up System (Promega). After inactivation of the restriction enzymes by incubation for 20 min at 658C, one-sixth of the digested PCR product was ligated with 25 ng of the SgfI-and PmeI-digested pF1K T7 vector in a 10-mL reaction of Flexi w Ligase buffer with 10 units of T4 DNA Ligase (Promega) for 60 min at 258C. One microliter of the ligation reaction was used to transform 10 mL of JM109 Competent Cells (.10 8 cfu/mg, Promega), and the transformants were selected on LB agar plates containing 50 mg/mL of kanamycin. A randomly chosen clone was first examined using the CloneChecker TM System (Invitrogen, Carlsbad, USA) by estimating the size of the supercoiled plasmid DNA. For ORFs cloned by the PCR cloning method, entire sequences were determined with the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, USA), with sequencing primers designed every approximate 400 bp in a single strand of ORF. The sequence data were obtained with ABI PRISM 3700 or 3130, and analyzed with SEQUENCHER TM sequence assembly software (Gene Codes Co., Ann Arbor, USA). For ORF Trap cloning, the ORF of interest was transferred to pF1Kof vector using a homologous recombination system in E. coli (JC8679), according to the method previously described. 9 For Flexi w Vector cloning using the pENT entry clones we had previously constructed, 9 the ORF of interest was recovered by digestion with BstBI and SnaBI restriction endonucleases whose recognition sites were located upstream and downstream of the ORF, respectively, in the vector portion. It was then inserted between the BstBI and SnaBI sites of the pSP73Flexi-1 vector, which contains SgfI and PmeI sites upstream and downstream of the BstBI and SnaBI sites, respectively. After cloning the ORFs into the pSP73Flexi-1 vector, they were cut with SgfI and PmeI restriction endonucleases and then recloned between the SgfI and PmeI sites of the pF1K vector.

In vitro protein production
For in vitro translation systems using Wheat Germ Extract Plus (WGEP) (Promega) and a TransDirect insect cell lysate (TD) (Shimadzu, Japan), RNA synthesis was performed using the T7 RiboMAX Express Large Scale RNA Production System (Promega) with linearized template DNAs, according to the supplier's instructions. After purification with the RNeasy Mini Kit (QIAGEN, Hilden, Germany), RNAs were concentrated by ethanol precipitation and dissolved in 15 mL of DEPC-treated water. In vitro translation reactions were performed in a reaction volume of 10 mL using 2.4 and 3.2 mg of the RNA for the WGEP and the TD systems, respectively, following the supplier's recommendation unless otherwise stated. 2.4. Examination of the effects of the flanking sequences of ORF on in vitro production of luciferase, Venus, and HaloTag For in vitro luc2 production, 1.4 mg of RNAs were used for WGEP and 3.8 mg for TD in a reaction volume of 12 mL. The activities of luc2 in an aliquot of the reaction mixture (10 mL) were assayed using the Dual-Glo Luciferase Assay System (Promega) according to the supplier's instructions. Luminescence was detected in a GloMAX 96 Microplate Luminometer (Promega). In vitro syntheses of Venus proteins were performed in 20 mL using 4.8 and 6.4 mg of the template RNA in the WGEP and the TD systems, respectively, after removing turbidity by centrifugation at 15 000 rpm for 2 min. Fluorescent signals were detected in the Applied Biosystems 7500 Fast Real-Time PCR System after 2 and 5 h reactions in the WGEP and TD systems, respectively. HaloTag-TMR ligand binding was monitored for quantification of the active HaloTag protein. Two microliters of 5 mM HaloTag-TMR ligand was added to an 8-mL reaction mixture, and the mixture was incubated at 258C for 5 min. The binding reaction was stopped by adding 2 mL of 6Â Laemmli's SDS sample buffer and then boiling at 1008C for 5 min. The solution (7.5 mL) was analyzed on 12.5% SDS -PAGE. Fluorescent signals from TMR were detected and quantified with FLA-3000 (Fujifilm, Tokyo, Japan) and MultiGauge image analyzing software (Fujifilm).

Western blotting
For Western blot analysis of HaloTag-fusion proteins, the proteins separated by SDS -PAGE were electrophoretically transferred onto a PVDF membrane (FluoroTrans W; PALL, Portsmouth, UK) using the transfer buffer [25 mM Tris -Cl ( pH 8.3), 192 mM glycine, 20% (V/V) methanol] with the BIOCRAFT BE-300 semidry transfer device (Biocraft, Tokyo, Japan). HaloTag-fusion proteins were detected as follows. The resultant PVDF membrane was washed with TBS [20 mM Tris -Cl ( pH 7.5) and 150 mM NaCl] including 0.05% Tween-20 (TBST) for 10 min with gentle agitation. After pre-incubation of the membrane with TBST containing 5% skim milk for 60 min, the membrane was incubated with 1:30 000 rabbit anti-HaloTag IgG antibody (Promega) in TBST containing 1% skim milk for 60 min. After washing with TBST for 5 min four times, the membrane was further incubated with 1:30 000 horseradish-peroxidase-conjugated anti-rabbit IgG antibody (Promega) in TBST containing 1% skim milk for 60 min. After washing the membrane with TBST, HaloTag-fusion proteins were finally detected using ECL plus (GE Healthcare) according to the manufacturer's instructions. The luminescent images were recorded by a Luminescent Image Analyzer LAS3000 (Fujifilm). MagicMark XP Western Protein Standard (Invitrogen) was used for estimation of the apparent molecular masses of the HaloTag-fusion proteins.

Examination of the subcellular localization of
HaloTag-fusion proteins The human embryonic kidney (HEK) 293 cell line was obtained from HSRRB of the Japan Health Sciences Foundation. HEK293 and COS-7 cell lines were grown in DMEM supplemented with 10% of Tet System Approved Fetal Bovine Serum (BD) and 1Â Antibiotic -Antimycotic reagent (100 U/mL penicillin þ 100 mg/mL streptomycin þ 0.25 mg/mL amphotericin B; Invitrogen). HEK293 and COS-7 were transfected with expression clones using FuGENE6 Transfection Reagent (Roche Diagnostics, Basel, Switzerland) in an 8-well chambered coverglass (Thermo Fisher Scientific, Rochester, USA) according to the supplier's instructions. Cells (200 mL, 4 Â 10 4 ) were plated 24 h before transfection. After 16-24 h of transfection, HaloTag-fusion proteins and DNA were labeled with medium with 1 mM of HaloTag-TMR ligand and Hoechst (33342, 3.3 mg/ mL, final concentration; Sigma-Aldrich, St. Louis, USA) for 15 min in a CO 2 (5%) incubator at 37ºC. After washing four times with 300 mL of medium, the cells were incubated for 0.5 -24 h in a CO 2 incubator and the subcellular localization of HaloTag-or MGFP-fusion proteins were observed with a Biozero fluorescence microscopy system (Keyence, Osaka, Japan) with 605/55 and 535/50 nm filters. Photos labeled with Hoechst33342 were merged with those of MGFP-or HaloTag-fusion proteins and the subcellular localizations of MGFP-and HaloTagfusion proteins were compared.

Construction of Flexi ORF clones
Led by the motivation described in Section 1, we have initiated preparation of Flexi w Vector ORF clones using human cDNA clones accumulated by the Kazusa cDNA project. 14 Table S1). These Flexi ORF clones contained relatively large ORFs derived from 1163 KIAA and 766 known genes, the average sizes of which are 2.8 and 2.4 kb, respectively. ORFs of 341 pF1K clones were PCR-cloned using PrimeStar HS or KOD plus high-fidelity DNA polymerases, as described in Section 2. All ORF clones obtained by PCR cloning were sequence-verified and we found that the PCR mutation occurrence rate was less than 5 Â 10 25 mutations per residue under the conditions employed in this study. ORF clones with large ORFs are inevitably at higher risk of having artificial missense mutation(s) introduced by PCR, thus we previously developed and applied the ORF Tap cloning method for the construction of Flexi ORF clones for some relatively long ORFs (mainly those .2 kb). 9 The method is based on a homologous recombination in E. coli JC8679 and thus is very unlikely to introduce mutation(s) into ORFs during cloning. We therefore considered that this method was also highly suited to the preparation of Flexi ORF clones because reconfirmation of the ORF sequence could be avoided. 16 -18 Within this context, 842 pF1K clones were constructed by the ORF Trap cloning method. The remaining 749 pF1K clones, termed Flexi_RBS type, were constructed by transferring ORF sequences from the Gateway entry clones we had previously prepared to the pF1K vector, because the 5 0 -and 3 0 -untranslated sequences (UTRs) have been already trimmed out in the Gateway entry clones. These clones contained a 19bp fragment, including a ribosome-binding site (RBS), between the SgfI site and a translational initiation codon. These Flexi clones were distinguished from others by adding 'SD', representing a Shine-Dalgarno sequence as an indication of the presence of an RBS, to their clone IDs, whereas the other pF1K clones were simply termed Flexi type. The ORF flanking sequences in these Flexi clones are shown in Fig. 1A. The SgfI site was placed one base upstream of the initiation codon, which allowed the production of recombinant proteins with the native translational initiation site or with N-terminal tags using appropriate Flexi w vectors. On the other hand, the PmeI site was placed just downstream from the original stop codon of the ORF, which resulted in an attachment of the Val in Flexi type or the Tyr-Val -Val in Flexi_RBS type to the carboxy end of the native ORF. When an ORF sequence flanked by SgfI and PmeI was cloned between the SgfI and EcoICRI sites of a Flexi w vector, the translational stop codon was destroyed and thus the protein was expressed as a carboxy-terminal fusion. ORFs in pF1K clones thus prepared can be easily transferred to other types of Flexi w vectors following digestion with SgfI and PmeI (Fig. 1B).
Information about the Flexi ORF clones (i.e. ORF nucleotide sequences, the predicted primary sequences of the gene products, corresponding Ensembl gene ID, and gene description 2 in addition to results of computer-assisted analyses of the predicted primary amino acid sequences) is available through our KOP database (http://www.kazusa.or.jp/ kop/).

Comparison of the productivities of HaloTag-
fusion proteins from Flexi ORF clones and Gateway ORF clones in two in vitro protein production systems Since our focus is on the use of HaloTag w technology, we initially prepared a set of Flexi ORF clones as HaloTag-fusions using the Flexi w cloning method. To examine the utility of HaloTag w technology, HaloTag-fusion products were produced in in vitro translation systems. The expressed proteins were analyzed on SDS -PAGE after labeling with a HaloTag-TMR ligand. This simple labeling procedure enabled us to fluorometrically detect only proteins carrying a HaloTag portion on SDS -PAGE, and thereby made it easy to check whether the ORF encoded an appropriately sized protein. Using this analysis, quantities of HaloTag-fusion proteins with ligand-binding activity could be directly estimated from the fluorescence intensities of protein bands on SDS -PAGE. In these experiments, we also compared the performance of two in vitro translation systems: one derived from a wheat germ extract and the second from an insect cell extract [commercially named WGEP and TransDirect (TD), respectively]. We used these systems because these eukaryotic cell extracts are known to produce larger amounts of proteins than conventional rabbit reticulocyte lysates. In parallel, we also compared the efficiency of protein synthesis by Gateway-type ( pTD2-Gw) and Flexi-type ( pTD2-Flx) expression constructs, where ORFs were flanked with different appended sequences that are associated with the respective cloning methods. These appended sequences may affect the translational activity of the HaloTag-fusion constructs in vitro. For this purpose, we constructed 13 sets of HaloTagfusion protein expression plasmids using pTD2-Gw

No. 3]
T. Nagase et al. and pTD2-Flx vectors. pTD2-Flx is a simple Flexi-type HaloTag-fusion protein expression vector derived from the pTD1 vector for the TD system. 19 This vector has 8-bp restriction enzyme recognition sites (SgfI and PmeI sites) flanked by the baculovirus polyhedrin 5 0 -and 3 0 -UTRs, respectively. pTD2-Gw is a hybrid expression vector derived from pTD2-Flx, which has 25-bp Gateway recombination sites, attB1 or attB2, between polyhedrin 5 0 -UTR and SgfI, or between PmeI and 3 0 -UTR, respectively ( Fig. 2A). In both systems, protein production was directed by invitro-synthesized RNAs. Many fluorescent bands, probably resulting from proteolytic degradation, were observed in most samples. The fluorescence signal intensities of the largest bands were the expected size for the respective protein products, and each HaloTag-fusion protein was recorded for comparing the protein productivities of pTD2-Flx and pTD2-Gw (Fig. 2B, indicated by an arrow). In the TD system, the fluorescence intensities of 8 out of 13 HaloTag-fusion proteins in the pTD2-Gw format were less than 60% as intense as those derived from pTD2-Flx clones (Fig. 2B, upper lanes:  2, 3, 5, 8-11, and 13). On the other hand, in the WGEP expression system, the fluorescence intensities of HaloTag-fusion proteins from five fusion constructs in the pTD2-Gw vector were slightly reduced compared with those from pTD2-Flx fusion constructs (Fig. 2B, lower lanes: 3, 4, 8-10). Interestingly enough, the fluorescent band patterns of HaloTagfusions produced in the TD and WGEP systems were considerably different from each other, although the same in-vitro-synthesized RNAs were subjected to both in vitro protein production systems. These results indicated that (1) the protein productivity of both the TD and WGEP systems varied widely from gene to gene, (2) TD and WGEP had different preferences for genes in terms of protein production, and (3) the Gateway recombination site(s) apparently had an inhibitory effect on translational activity, which was more prominent in the TD systems.
3.3. Effects of ORF-flanking sequences on translational activity in cell-free protein synthesis systems While the different performances of the TD and WGEP systems were anticipated to some extent, it was not anticipated that the Gateway recombination sites would have an inhibitory effect on in vitro protein production. Thus, we further pursued the cause of this apparent inhibitory effect of the Gateway recombination sites on in vitro protein production. To systematically examine the effects of the appended sequences flanking the ORFs on in vitro protein production, we constructed systematically modified versions of expression clones for luc2, HaloTag, and Venus autofluorescent proteins, with different flanking sequences for each ORF (Fig. 2C). Protein production in the TD and WGEP systems was monitored by measuring luciferase activity, HaloTag-TMR ligand-binding activity, or the fluorescence intensity of Venus protein (Fig. 2D). As shown in Fig. 2C, these clones included flanking sequences derived from the Gateway attB sites (B1 and/or B2), or parts of the kanamycin-resistant gene (K1: ACTGGGCACAACAGACAATCGGCTG, or K2: CCTGTCATCTCACCTTGCTCCTGCC, as examples of non-specific sequences) between the translational enhancer ( polyhedrin 5 0 -UTR) and the initiation ATG codon, and/or between the stop codon and the polyhedrin 3 0 -UTR. N -N, an expression clone without any additional sequences, was the control clone. N -B2 and N -K2 carry additional sequences (B2 and K2, respectively) only at the 3 0 -flanking site, whereas B1 -N and K1 -N have additional sequences (B1 and K1, respectively) only at the 5 0 -flanking site. K1 and K2 were spacer sequences with equal sizes as B1 and B2 and were not expected to be involved with the Gateway recombination. We also compared protein production from the Gateway-type expression vector (B1-B2) and the modified vector (B1-K2) in parallel. In total, we examined seven different expression vectors in these experiments. Figure 2D shows the relative activities of luc2, HaloTag, and Venus proteins produced in the TD and WGEP systems compared with the N -N type of pTDFlexi vector. Under these experimental conditions, the luciferase expression clones with attB1 sequence upstream of the ORFs (Fig. 2, B1-N, B1 -B2, and B1 -K2) appeared to exhibit only a slight inhibitory effect in the TD and WGEP systems in comparison with the pTDFlexi-luc2 construct (N -N in Fig. 2C). This reduction in protein productivity seemed to depend on the distance (39 nucleotides) between the 5 0 translational enhancer and the initiation ATG but not the specific nucleotide sequence, at least in insect TD systems. Expression clones with short nucleotide sequences of the same length derived from the kanamycin-resistant gene also reduced the production of luciferase (Fig. 2D, upper,  K1-N). Such an inhibitory effect was more evident for HaloTag and Venus protein production in the TD system. In fact, in the TD system, both HaloTag-TMRligand-binding activity and the fluorescence intensity of Venus decreased to less than 50% when the additional sequences were located upstream of the ORF (Fig. 2D, upper, B1 -N, K1-N, B1 -B2, and B1 -K2). These results indicated that protein productivity in the TD system was affected by the increase in distance between the polyhedrin 5 0 -UTR and the start ATG, rather than by the flanking nucleotide sequence of the spacer.
It was reported that the polyhedrin 5 0 -UTR in pTD1 displayed a strong translational enhancing activity, even in the wheat germ extract and rabbit reticulocyte lysate systems. 19 The inhibitory effect of sequences upstream of the ORF was prominent in the TD system but only minor in the WGEP system (Fig. 2D, lower). Our results also showed that the expression clone lacking 5 0 -UTR considerably reduced the productivity of luciferase in both the TD and WGEP systems, indicating that the translational enhancer of 5 0 -UTR in pTD1 was functional in each system (Fig. 2D, D5). On the other hand, translational activity was largely unaffected by the spacing between the 3 0 -translational enhancer and the translational stop codon in all experiments using luc2, HaloTag, and Venus proteins (Fig. 2D, N -B2, N -K2). Taking all these results together, we concluded that the spacing between the 5 0 -translational enhancer and the translational initiation codon affected protein productivity, at least in vitro. Although the mechanism of the inhibitory effect on translational activity by spacing remains to be elucidated, it appears that nucleotide spacing between the enhancer and the initiation codon can sometimes affect translational activity. Because the distance between the 5 0 -translational enhancer and the initiation codon is shorter in the Flexi ORF clone than in the Gateway ORF clone, these results suggest that the Flexi ORF clone could be the clone of choice to consistently obtain high protein productivity in both the TD and WGEP systems. We consider this as another benefit of using the Flexi ORF clones, since production level is a serious concern, particularly when a recombinant protein is synthesized in vitro.

Quality check of ORF clones from the viewpoint of
in vitro protein production Based on the results described above, we confirmed the functionality of a number of Flexi ORF clones produced as HaloTag-fusions in in vitro protein production systems. For this purpose, we used a transcription/translation coupled wheat-germ-cellfree protein synthesis system [TNT SP6 High Yield Protein Expression System, hereafter abbreviated as (TNT SP6 HYPES)] because the coupled system is easier and faster to operate and gives a higher yield of recombinant protein production than other uncoupled cell-free protein synthesis systems. Eight hundred and fourteen ORF sequences in pF1K clones were transferred to pF3AHT, C-terminal HaloTag-fusion protein expression vectors, by Flexi w cloning methods. One practical problem we encountered was that the coupled system required a large amount of expression plasmids to obtain the best results. Since we planned to use a small volume of E. coli culture (about 1 mL) to produce the plasmids, we were concerned that we would not be able to produce enough DNA to obtain good results in the TNT system. To solve this problem, we took advantage of in vitro amplification of expression plasmids. We found that DNA amplified with a TempliPhi rolling circle amplification kit could be used as template DNA in the TNT SP6 HYPES without any purification. 20 This enabled us to examine the production of a number of HaloTag-fusions in a high-throughput manner. As a result, medium-sized and even relatively large proteins, composed of more than 1000 amino acid residues, were effectively produced and detected by Western blot analysis using anti-HaloTag IgG, with only small amounts of degraded or inappropriately initiated proteins (Fig. 3A). Moreover, most of the HaloTag-fusion proteins retained HaloTag ligandbinding activities (Fig. 3B). Of the 814 pF3AHT clones tested in the TNT system, 97% produced recombinant proteins that could be detected by TMR HaloTag ligand labeling and Western blot analysis using anti-HaloTag antibody. The apparent molecular masses of the recombinant proteins were estimated by SDS -PAGE followed by Western blot analysis using anti-HaloTag antibodies. Most of the proteins had apparent molecular masses that were consistent with the numbers of amino acid residues of the fused proteins (data not shown). Although the fluorescence intensity of TMR varied widely from protein to protein in a range of arbitrary fluorescence units from 10 to 10 000, HaloTag-fusions that retained the functional characteristics of TMR ligand-binding were efficiently produced. However, we did observe a tendency of HaloTag-TMR ligand binding to display decreases in fluorescence intensity with increases in protein size (Fig. 3C). These results are most likely explained by decreases in the production levels of larger HaloTag-fusions. However, it could also be explained on the assumption that the partner proteins may affect HaloTag-TMR ligandbinding activities. To address this, we examined the relationship between protein yields and their HaloTag ligand-binding activities for 53 HaloTagfusions. In this experiment, the HaloTag-fusion protein yields were expressed by the radioactivity of [ 35 S]methionine and [ 35 S]cysteine incorporated into the proteins and divided by the number that contained methionine and cysteine, while the HaloTag ligand-binding activities were estimated from the fluorescence intensity of Cy5-labeled HaloTag ligandbound HaloTag-fusion proteins on SDS -PAGE. The HaloTag-fusion protein yields and the HaloTag ligand-binding activities are plotted against the number of their amino acid residues in Fig. 3D and E, respectively. In Fig. 3F, the ratios of the HaloTag ligand-binding activities to the yields of the HaloTagfusion proteins are plotted against the number of amino acid residues. The results indicated that the molecular HaloTag ligand-binding activities did not vary widely from fusion to fusion. Based on this result, we consider that the low ligand-binding activities observed resulted from low production levels of HaloTag-fusion proteins, although there may have been some exceptions.

Subcellular localization of HaloTag-fusion proteins
and MGFP-fusion proteins In general, information on the subcellular localization of proteins provides us with an important clue on potential protein function. Thus, we accumulated data of the subcellular localizations using green fluorescent protein (GFP) -ORF fusions constructed using the Gateway system. It was important for us to No. 3] T. Nagase et al.
do this, we prepared pFC8A expression clones for the HaloTag protein containing signal sequences, such as membane-localized signal (Memb), 21 nuclear localization signal (NLS), 22 endoplasmic reticulum targeting sequence (ER), 23 Golgi-localized signal (Golgi), 24 and mitochondrial targeting sequence (Mito). 25 These sequences were all placed at the N-terminal region of the HaloTag proteins. Eighteen hours after transfection of these constructs into COS-7 cells, the HaloTagfusion proteins were labeled with the TMR ligand and observed with a fluorescence microscopy system. Figure 4A shows that all fused proteins were detected in an expected subcellular compartment, indicating that HaloTag proteins do not inhibit the function of these subcellular localization signals. Next, we directly evaluated whether HaloTag-fusion proteins were colocalized with the same GFP-fusion proteins when both fusions were expressed in the same cells. C-terminal HaloTag-and MGFP-fusion protein expression clones were prepared by the Flexi w cloning system using the Flexi ORF clones we prepared and pFC8A (HaloTag) and pFC8A (MGFP) vectors. After co-transfection of HEK293 cells with the same ORF clone fused to the two different fusion tags, HaloTagfusion proteins were labeled with the TMR ligand and then the fluorescence images were obtained for TMR-(red) and MGFP-labeled (green) proteins for 40 ORFs. Examples of the cellular localization of the fusion proteins are shown in Fig. 4B. Among them, subcellular locations of glucocorticoid modulatory element-binding protein 2 (GMEB-2/KIAA1269), phosphatidylserine synthase 1 (PTDSS1/KIAA0024), membrane-bound transcription factor site-1 protease (MBTPS1/KIAA0091), and transmembrane protein 127 (TMEM127/KIBB2508) are reported in the UniProt Knowledgebase as nucleus/cytoplasm, membrane, endoplasmic reticulum/Golgi apparatus, and membrane, respectively (http://ca.expasy.org/ sprot/). 26 In contrast, the subcellular location of Mesoderm development candidate 2 (MESDC2/ KIAA0081) is not mentioned and Secernin-1 (SCRN1/KIAA0193) is predicted to exist in the cytoplasm from similarities found in the database. The subcellular localizations of the 40 HaloTag-fusion proteins analyzed here were the same as those for MGFP-fusion proteins. These results indicate that HaloTag-fusion proteins can be used as an alternative for subcellular localization analysis in place of conventional autofluorescent proteins.
In conclusion, a complete set of ORF resources in various expression-ready clone formats clearly can serve as versatile sets of reagents for functional genomics research. The rapid, easy, and assured transfer of ORF sequences into different expression vectors is highly critical for this purpose, since many ORF clones are used in combination with protein fusion tags that allow functional analysis. Within this context, we selected a HaloTag protein as a fusion partner because we considered that HaloTag w technology would provide a wide range of utilities that include detection and purification as well as adaptability in functional studies. In fact, we have successfully used HaloTag-fusion proteins in other studies for pulse-chase labeling, protein -protein interaction, and chromosomal immunoprecipitation-like analyses along with bioimaging (unpublished data). In this report, we have shown that the Flexi w cloning method is suitable for the preparation of a large number of ORF clones into the pF1K vector and allows seamless implementation of HaloTag w interchangeable technology. Moreover, we have demonstrated that most human proteins were efficiently expressed as HaloTag-fusions in an in vitro coupled protein expression system, as well as in cultured mammalian cells. The resultant HaloTagfusion proteins were biochemically active and successfully used for analyses of subcellular localization as an alternative to conventional autofluorescent proteins. Based on these results, we will accumulate a number of human ORF clones in the form of expression plasmids for HaloTag-fusion proteins, either C-or N-terminal fusions, in our ORFeome project since they will be directly used for functional studies, as well as for further transfer to another vector. HaloTag w technology provides a single fusion tag that can be utilized for a variety of structural and functional proteomic studies. Although there are a number of ORFeome resources for human genes that have been generated by Gateway recombinational cloning, 27 a set of human Flexi ORF clones prepared as described would provide the research community with an important alternative for largescale proteomic studies and a tool for individual research laboratories.