NMR-Guided Repositioning of Non-Steroidal Anti-Inflammatory Drugs into Tight Junction Modulators.

Bioavailability is a major bottleneck in the clinical application of medium molecular weight therapeutics, including protein and peptide drugs. Paracellular transport of these molecules is hampered by intercellular tight junction (TJ) complexes. Therefore, safe chemical regulators for TJ loosening are desired. Here, we showed a potential application of select non-steroidal anti-inflammatory drugs (NSAIDs) as TJ modulators. Based on our previous observation that diclofenac and flufenamic acid directly bound various PDZ domains with a broad specificity, we applied solution nuclear magnetic resonance techniques to examine the interaction of other NSAIDs and the first PDZ domain (PDZ1) of zonula occludens (ZO)-1, ZO-1(PDZ1). Inhibition of ZO-1(PDZ1) is expected to provide loosening of the epithelial barrier function because the domain plays a crucial role in maintaining TJ integrity. Accordingly, diclofenac and indomethacin were found to decrease the subcellular localization of claudin (CLD)-2 but not occludin and ZO-1 at the apicolateral intercellular compartment of Madin-Darby canine kidney (MDCK) II cells. These NSAIDs exhibited 125-155% improved paracellular efflux of fluorescein isothiocyanate insulin for the Caco-2 cell monolayer. We propose that these NSAIDs can be repurposed as drug absorption enhancers for peptide drugs.

Opposing Effect of Naringenin and Quercetin on the Junctional Compartment of MDCK II Cells to Modulate the Tight Junction.

Maintaining tight junction (TJ) integrity is important for epithelial cell barriers. Previously, the enhancement of TJ integrity, induced by citrus-derived flavonoids, naringin (NRG) and hesperidin (HSD), was demonstrated, but the effects of their aglycones naringenin (NAR) and hesperetin (HST), and the mechanisms, have not been systematically investigated. Here we compared three series of flavonoids related to NAR, HST, quercetin (QUE) and their glycosides with the Madin-Darby canine kidney (MDCK) II cell monolayers. The effect of flavonoids on the protein expression level of claudin (CLD)-2 and its subcellular localization were investigated. NAR, NRG, and HSD increased the CLD-2 localization at the TJ compartment, and its protein expression level. QUE and HST showed TJ-mitigating activity. Narirutin (NRT), neohesperidin (NHD) and rutin (RUT) did not affect the TJ. In addition, NAR and QUE induced an increase or decrease of the transepithelial electrical resistance (TEER) values of the MDCK II monolayers. Two known signaling pathways, phosphatidyl-inositol-3 kinase (PI3K) and 5'-AMP-activated protein kinase (AMPK), were further compared with NAR. Two-dimensional polyacrylamide electrophoresis (2D PAGE) analysis of whole-cell proteins treated with NAR, AICA-riboside (AMPK activator) and LY294002 (PI3K inhibitor) showed in both a distinct pattern. This suggests the target of NAR's CLD-2 or zonula occludens-1 (ZO-1) modulation was unique.

Discovery of Potent Disheveled/Dvl Inhibitors Using Virtual Screening Optimized With NMR-Based Docking Performance Index.

Most solid tumors have their own cancer stem cells (CSCs), which are resistant to standard chemo-therapies. Recent reports have described that Wnt pathway plays a key role in self-renewal and tumorigenesis of CSCs. Regarding the Wnt/ß-catenin pathway, Dvl (mammalian Disheveled) is an attractive target of drug discovery. After analyzing the PDZ domain of human Dvl1 (Dvl1-PDZ) using NMR, we subjected it to preliminary NMR titration studies with 17 potential PDZ-binding molecules including CalBioChem-322338, a commercially available Dvl PDZ domain inhibitor. Next, we performed virtual screening (VS) using the program GOLD with nine parameter sets. Results were evaluated using the NMR-derived docking performance index (NMR-DPI). One parameter set of GOLD docking showing the best NMR-DPI was selected and used for the second VS against 5,135 compounds. The second docking trial identified more than 1,700 compounds that exhibited higher scores than CalBioChem-322338. Subsequent NMR titration experiments with five new candidate molecules (NPL-4001, 4004, 4011, 4012, and 4013), Dvl1-PDZ revealed larger chemical shift changes than those of CalBioChem-322338. Finally, these compounds showed partial proliferation inhibition activity against BT-20, a triple negative breast cancer (TNBC) cell. These compounds are promising Wnt pathway inhibitors that are potentially useful for anti-TNBC therapy.

Spatial Overlap of Claudin- and Phosphatidylinositol Phosphate-Binding Sites on the First PDZ Domain of Zonula Occludens 1 Studied by NMR.

Background: The tight junction is an intercellular adhesion complex composed of claudins (CLDs), occludin, and the scaffolding proteins zonula occludens 1 (ZO-1) and its two paralogs ZO-2 and ZO-3. ZO-1 is a multifunctional protein that contains three PSD95/Discs large/ZO-1(PDZ) domains. A key functional domain of ZO-1 is the first PDZ domain (ZO-1(PDZ1)) that recognizes the conserved C-termini of CLDs. Methods: In this study, we confirmed that phosphoinositides bound directly to ZO-1(PDZ1) by biochemical and solution NMR experiments. We further determined the solution structure of mouse ZO-1(PDZ1) by NMR and mapped the phosphoinositide binding site onto its molecular surface. Results: The phosphoinositide binding site was spatially overlapped with the CLD-binding site of ZO-1(PDZ1). Accordingly, inositol-hexaphosphate (phytic acid), an analog of the phosphoinositide head group, competed with ZO-1(PDZ)-CLD interaction. Conclusions: The results suggested that the PDZ domain⁻phosphoinositide interaction plays a regulatory role in biogenesis and homeostasis of the tight junction.

Discovery of Cryoprotective Activity in Human Genome-Derived Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are an emerging phenomenon. They may have a high degree of flexibility in their polypeptide chains, which lack a stable 3D structure. Although several biological functions of IDPs have been proposed, their general function is not known. The only finding related to their function is the genetically conserved YSK2 motif present in plant dehydrins. These proteins were shown to be IDPs with the YSK2 motif serving as a core region for the dehydrins' cryoprotective activity. Here we examined the cryoprotective activity of randomly selected IDPs toward the model enzyme lactate dehydrogenase (LDH). All five IDPs that were examined were in the range of 35-45 amino acid residues in length and were equally potent at a concentration of 50 µg/mL, whereas folded proteins, the PSD-95/Dlg/ZO-1 (PDZ) domain, and lysozymes had no potency. We further examined their cryoprotective activity toward glutathione S-transferase as an example of the other enzyme, and toward enhanced green fluorescent protein as a non-enzyme protein example. We further examined the lyophilization protective activity of the peptides toward LDH, which revealed that some IDPs showed a higher activity than that of bovine serum albumin (BSA). Based on these observations, we propose that cryoprotection is a general feature of IDPs. Our findings may become a clue to various industrial applications of IDPs in the future.

Nuclear magnetic resonance evidence for the dimer formation of beta amyloid peptide 1-42 in 1,1,1,3,3,3-hexafluoro-2-propanol.

Alzheimer's disease involves accumulation of senile plaques in which filamentous aggregates of amyloid beta (Aß) peptides are deposited. Recent studies demonstrate that oligomerization pathways of Aß peptides may be complicated. To understand the mechanisms of Aß(1-42) oligomer formation in more detail, we have established a method to produce (15)N-labeled Aß(1-42) suited for nuclear magnetic resonance (NMR) studies. For physicochemical studies, the starting protein material should be solely monomeric and all Aß aggregates must be removed. Here, we succeeded in fractionating a "precipitation-resistant" fraction of Aß(1-42) from an "aggregation-prone" fraction by high-performance liquid chromatography (HPLC), even from bacterially overexpressed Aß(1-42). However, both Aß(1-42) fractions after 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) treatment formed amyloid fibrils. This indicates that the "aggregation seed" was not completely monomerized during HFIP treatment. In addition, Aß(1-42) dissolved in HFIP was found to display a monomer-dimer equilibrium, as shown by two-dimensional (1)H-(15)N NMR. We demonstrated that the initial concentration of Aß during the HFIP pretreatment altered the kinetic profiles of Aß fibril formation in a thioflavin T fluorescence assay. The findings described here should ensure reproducible results when studying the Aß(1-42) peptide.

A Method for Systematic Assessment of Intrinsically Disordered Protein Regions by NMR.

Intrinsically disordered proteins (IDPs) that lack stable conformations and are highly flexible have attracted the attention of biologists. Therefore, the development of a systematic method to identify polypeptide regions that are unstructured in solution is important. We have designed an "indirect/reflected" detection system for evaluating the physicochemical properties of IDPs using nuclear magnetic resonance (NMR). This approach employs a "chimeric membrane protein"-based method using the thermostable membrane protein PH0471. This protein contains two domains, a transmembrane helical region and a C-terminal OB (oligonucleotide/oligosaccharide binding)-fold domain (named NfeDC domain), connected by a flexible linker. NMR signals of the OB-fold domain of detergent-solubilized PH0471 are observed because of the flexibility of the linker region. In this study, the linker region was substituted with target IDPs. Fifty-three candidates were selected using the prediction tool POODLE and 35 expression vectors were constructed. Subsequently, we obtained 15N-labeled chimeric PH0471 proteins with 25 IDPs as linkers. The NMR spectra allowed us to classify IDPs into three categories: flexible, moderately flexible, and inflexible. The inflexible IDPs contain membrane-associating or aggregation-prone sequences. This is the first attempt to use an indirect/reflected NMR method to evaluate IDPs and can verify the predictions derived from our computational tools.

Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-ß assembly.

Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid ß-protein (Aß) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aß oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na(+)/K(+)-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aß-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.

An optimized Npro-based method for the expression and purification of intrinsically disordered proteins for an NMR study.

Intrinsically disordered proteins (IDPs) are an emerging concept. IDPs have high flexibility in their polypeptide chains, lacking a stable 3-dimensional structure. Because of the difficulty in performing X-ray crystallography for IDPs, nuclear magnetic resonance (NMR) spectroscopy is the first choice for atomic-level investigation of their nature. Given that isotopically labeled IDP samples are necessary for NMR study, a robust and cost-effective protocol for bacterial expression and purification of IDP is also needed. We employed the Npro (EDDIE)-autoprotease fusion protein system. Although IDPs are believed to be readily degraded by endogenous proteases when expressed in Escherichia coli, Npro-fused IDPs showed excellent resistance to degradation. Seven IDPs of uncharacterized function sampled from the human genome as well as 3 constructs from IDP regions derived from human FancM and Thermococcus kodakarensis Hef were prepared. We improved the protocol of refolding of Npro (EDDIE) to use dialysis, which is convenient for subsequent purification using reversed-phase (RP) HPLC. The method is robust and widely applicable to any IDP sample, promoting the acquisition of experimental data for IDPs in a high-throughput manner.

Effect of Ca2+ on the microtubule-severing enzyme p60-katanin. Insight into the substrate-dependent activation mechanism.

Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca(2+) ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca(2+). In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca(2+) reduces elevated ATPase activity to the basal level. We identify the Ca(2+) -binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca(2+) regulates both severing and ATP hydrolysis activity, because the Ca(2+) -binding site on the N-terminal domain moves close to the AAA domain during MT severing.

¹H, ¹³C, and ¹5N resonance assignment of the SPFH domain of human stomatin.

Stomatin, a 288-residue protein, is a component of the membrane skeleton of red blood cells (RBCs), which helps to physically support the membrane and maintains its function. In RBCs, stomatin binds to the glucose transporter GLUT-1 and may regulate its function. Stomatin has a stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of its polypeptide chain. There are 12 SPFH domain-containing proteins, most of which are localized at the cellular or subcellular membranes. Although the molecular function of the SPFH domain has not yet been established, the domain may be involved in protein oligomerization. The SPFH domain of the archaeal stomatin homolog has been shown to form unique oligomers. Here we report the (15)N, (13)C, and (1)H chemical shift assignments of the SPFH domain of human stomatin [hSTOM(SPFH)]. These may help in determining the structure of hSTOM(SPFH) in solution as well as in clarifying its involvement in protein oligomerization.

Structure and function of the N-terminal nucleolin binding domain of nuclear valosin-containing protein-like 2 (NVL2) harboring a nucleolar localization signal.

The N-terminal regions of AAA-ATPases (ATPase associated with various cellular activities) often contain a domain that defines the distinct functions of the enzymes, such as substrate specificity and subcellular localization. As described herein, we have determined the solution structure of an N-terminal unique domain isolated from nuclear valosin-containing protein (VCP)-like protein 2 (NVL2(UD)). NVL2(UD) contains three α helices with an organization resembling that of a winged helix motif, whereas a pair of ß-strands is missing. The structure is unique and distinct from those of other known type II AAA-ATPases, such as VCP. Consequently, we identified nucleolin from a HeLa cell extract as a binding partner of this domain. Nucleolin contains a long (∼300 amino acids) intrinsically unstructured region, followed by the four tandem RNA recognition motifs and the C-terminal glycine/arginine-rich domain. Binding analyses revealed that NVL2(UD) potentially binds to any of the combinations of two successive RNA binding domains in the presence of RNA. Furthermore, NVL2(UD) has a characteristic loop, in which the key basic residues RRKR are exposed to the solvent at the edge of the molecule. The mutation study showed that these residues are necessary and sufficient for nucleolin-RNA complex binding as well as nucleolar localization. Based on the observations presented above, we propose that NVL2 serves as an unfoldase for the nucleolin-RNA complex. As inferred from its RNA dependence and its ATPase activity, NVL2 might facilitate the dissociation and recycling of nucleolin, thereby promoting efficient ribosome biogenesis.

Structural difference of vasoactive intestinal peptide in two distinct membrane-mimicking environments.

Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide which belongs to a glucagon/secretin superfamily, the ligand of class II G protein-coupled receptors. Knowledge for the conformation of VIP bound to membrane is important because the receptor activation is initiated by membrane binding of VIP. We have previously observed that VIP-G (glycine-extended VIP) is unstructured in solution, as evidenced by the limited NMR chemical shift dispersion. In this study, we determined the three-dimensional structures of VIP-G in two distinct membrane-mimicking environments. Although these are basically similar structures composed of a disordered N-terminal region and a long α-helix, micelle-bound VIP-G has a curved α-helix. The side chains of residues Phe(6), Tyr(10), Leu(13), and Met(17) found at the concave face form a hydrophobic patch in the micelle-bound state. The structural differences in two distinct membrane-mimicking environments show that the micelle-bound VIP-G localized at the water-micelle boundary with these side chains toward micelle interior. In micelle-bound PACAP-38 (one of the glucagon/secretin superfamily peptide) structure, the identical hydrophobic residues form the micelle-binding interface. This result suggests that these residues play an important role for the membrane binding of VIP and PACAP.

A common substrate recognition mode conserved between katanin p60 and VPS4 governs microtubule severing and membrane skeleton reorganization.

Katanin p60 (kp60), a microtubule-severing enzyme, plays a key role in cytoskeletal reorganization during various cellular events in an ATP-dependent manner. We show that a single domain isolated from the N terminus of mouse katanin p60 (kp60-NTD) binds to tubulin. The solution structure of kp60-NTD was determined by NMR. Although their sequence similarities were as low as 20%, the structure of kp60-NTD revealed a striking similarity to those of the microtubule interacting and trafficking (MIT) domains, which adopt anti-parallel three-stranded helix bundle. In particular, the arrangement of helices 2 and 3 is well conserved between kp60-NTD and the MIT domain from Vps4, which is a homologous protein that promotes disassembly of the endosomal sorting complexes required for transport III membrane skeleton complex. Mutation studies revealed that the positively charged surface formed by helices 2 and 3 binds tubulin. This binding mode resembles the interaction between the MIT domain of Vps4 and Vps2/CHMP1a, a component of endosomal sorting complexes required for transport III. Our results show that both the molecular architecture and the binding modes are conserved between two AAA-ATPases, kp60 and Vps4. A common mechanism is evolutionarily conserved between two distinct cellular events, one that drives microtubule severing and the other involving membrane skeletal reorganization.

Fine-tuning of protein domain boundary by minimizing potential coiled coil regions.

Structural determination of individual protein domains isolated from multidomain proteins is a common approach in the post-genomic era. Novel and thus uncharacterized domains liberated from intact proteins often self-associate due to incorrectly defined domain boundaries. Self-association results in missing signals, poor signal dispersion and a low signal-to-noise ratio in (1)H-(15)N HSQC spectra. We have found that a putative, non-canonical coiled coil region close to a domain boundary can cause transient hydrophobic self-association and monomer-dimer equilibrium in solution. Here we propose a rational method to predict putative coiled coil regions adjacent to the globular core domain using the program COILS. Except for the amino acid sequence, no preexisting knowledge concerning the domain is required. A small number of mutant proteins with a minimized coiled coil region have been rationally designed and tested. The engineered domains exhibit decreased self-association as assessed by (1)H-(15)N HSQC spectra with improved peak dispersion and sharper cross peaks. Two successful examples of isolating novel N-terminal domains from AAA-ATPases are demonstrated. Our method is useful for the experimental determination of domain boundaries suited for structural genomics studies.

The common phospholipid-binding activity of the N-terminal domains of PEX1 and VCP/p97.

PEX1 is a type II AAA-ATPase that is indispensable for biogenesis and maintenance of the peroxisome, an organelle responsible for the primary metabolism of lipids, such as beta-oxidation and lipid biosynthesis. Recently, we demonstrated a striking structural similarity between its N-terminal domain and those of other membrane-related AAA-ATPases, such as valosine-containing protein (p97). The N-terminal domain of valosine-containing protein serves as an interface to its adaptor proteins p47 and Ufd1, whereas the physiologic interaction partner of the N-terminal domain of PEX1 remains unknown. Here we found that N-terminal domains isolated from valosine-containing protein, as well as from PEX1, bind phosphoinositides. The N-terminal domain of PEX1 appears to preferentially bind phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate, whereas the N-terminal domain of valosine-containing protein displays broad and nonspecific lipid binding. Although N-ethylmaleimide-sensitive fusion protein, CDC48 and Ufd1 have structures similar to that of valosine-containing protein, they displayed lipid specificity similar to that of the N-terminal domain of PEX1 in the assays. By mutational analysis, we demonstrate that a conserved arginine surrounded by hydrophobic residues is essential for lipid binding, despite very low sequence similarity between PEX1 and valosine-containing protein.

Structural characterization of the MIT domain from human Vps4b.

The microtubule interacting and trafficking (MIT) domain is a small protein module of unknown function that is conserved in proteins of diverse function, such as Vps4, sorting nexin 15 (SNX15), and spastin. One non-synonymous single nucleotide polymorphism was reported, which results in a Ile58-to-Met (I58M) substitution in hVps4b. Here, we have determined the solution structure of the MIT domain isolated from the NH(2)-terminus of human Vps4b, an AAA-ATPase involved in multivesicular body formation. The MIT domain adopts an 'up-and-down' three-helix bundle. Comparison with the sequences of other MIT domains clearly shows that the residues involved in inter-helical contacts are well conserved. The Ile58-to-Met substitution resulted a substantial thermal instability. In addition, we found a shallow crevice between helices A and C that may serve as a protein-binding site. We propose that the MIT domain serves as a putative adaptor domain for the ESCRT-III complex involved in endosomal trafficking.

Crystallographic characterization of the N-terminal domain of PEX1.

Peroxisomal enzymes are responsible for several primary metabolism pathways, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases and both are necessary for the import of more than 50 peroxisomal resident proteins from the cytosol into peroxisomes. In this study, PEX1 N-terminal domain crystals have been prepared. The crystals belong to space group P3(1) or P3(2), with unit-cell parameters a = b = 63.5 A, c = 33.5 A, and contain one protein molecule per crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.05 A.

Structure of the N-terminal domain of PEX1 AAA-ATPase. Characterization of a putative adaptor-binding domain.

Peroxisomes are responsible for several pathways in primary metabolism, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases, both of which are indispensable in targeting over 50 peroxisomal resident proteins from the cytosol to the peroxisomes. Although the tandem AAA-ATPase domains in the central region of PEX1 and PEX6 are highly similar, the N-terminal sequences are unique. To better understand the distinct molecular function of these two proteins, we analyzed the unique N-terminal domain (NTD) of PEX1. Extensive computational analysis revealed weak similarity (<10% identity) of PEX1 NTD to the N-terminal domains of other membrane-related type II AAA-ATPases, such as VCP (p97) and NSF. We have determined the crystal structure of mouse PEX1 NTD at 2.05-A resolution, which clearly demonstrated that the domain belongs to the double-psi-barrel fold family found in the other AAA-ATPases. The N-domains of both VCP and NSF are structural neighbors of PEX1 NTD with a 2.7- and 2.1-A root mean square deviation of backbone atoms, respectively. Our findings suggest that the supradomain architecture, which is composed of a single N-terminal domain followed by tandem AAA domains, is a common feature of organellar membrane-associating AAA-ATPases. We propose that PEX1 functions as a protein unfoldase in peroxisomal biogenesis, using its N-terminal putative adaptor-binding domain.

High-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling.

Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost-effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for the preparation of DNA fragments were applied depending on the length of the peptides, such as polymerase chain reaction, chemical synthesis or a 'semi-synthetic method', which is a combination of chemical synthesis and enzymatic extension. The ability easily to construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in areas of biomedical research and drug discovery.