Cell-Free Synthesis of Human Endothelin Receptors and Its Application to Ribosome Display.

Engineering G-protein-coupled receptors (GPCRs) for improved stability or altered function is of great interest, as GPCRs consist of the largest protein family, are involved in many important signaling pathways, and thus, are one of the major drug targets. Here, we report the development of a high-throughput screening method for GPCRs using a reconstituted in vitro transcription-translation (IVTT) system. Human endothelin receptor type-B (ETBR), a class A GPCR that binds endothelin-1 (ET-1), a 21-residue peptide hormone, was synthesized in the presence of nanodisc (ND) composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The ET-1 binding of ETBR was significantly reduced or was undetectable when other phospholipids were used for ND preparation. However, when functional ETBR purified from Sf9 cells was reconstituted into NDs, ET-1 binding was observed with two different phospholipids tested, including POPG. These results suggest that POPG likely supports the folding of ETBR into its functional form in the IVTT system. Using the same conditions as ETBR, whose three-dimensional structure has been solved, human endothelin receptor type-A (ETAR), whose three-dimensional structure remains unsolved, was also synthesized in its functional form. By adding POPG-ND to the IVTT system, both ETAR and ETBR were successfully subjected to ribosome display, a method of in vitro directed evolution that facilitates the screening of up to 1012 mutants. Finally, using a mock library, we showed that ribosome display can be applied for gene screening of ETBR, suggesting that high-throughput screening and directed evolution of GPCRs is possible in vitro.

Bottom-up Creation of an Artificial Cell Covered with the Adhesive Bacterionanofiber Protein AtaA.

The bacterial cell surface structure has important roles for various cellular functions. However, research on reconstituting bacterial cell surface structures is limited. This study aimed to bottom-up create a cell-sized liposome covered with AtaA, the adhesive bacterionanofiber protein localized on the cell surface of Acinetobacter sp. Tol 5, without the use of the protein secretion and assembly machineries. Liposomes containing a benzylguanine derivative-modified phospholipid were decorated with a truncated AtaA protein fused to a SNAP-tag expressed in a soluble fraction in Escherichia coli. The obtained liposome showed a similar surface structure and function to that of native Tol 5 cells and adhered to both hydrophobic and hydrophilic solid surfaces. Furthermore, this artificial cell was able to drive an enzymatic reaction in the adhesive state. The developed artificial cellular system will allow for analysis of not only AtaA, but also other cell surface proteins under a cell-mimicking environment. In addition, AtaA-decorated artificial cells may inspire the development of biotechnological applications that require immobilization of cells onto a variety of solid surfaces, in particular, in environments where the use of genetically modified organisms is prohibited.

Antigen-responsive fluorescent antibody probes generated by selective N-terminal modification of IgGs.

Novel fluorescent antibody probes, which show antigen-dependent fluorescence responses, were developed by selective N-terminal fluorescent labeling of IgG monoclonal antibodies using a reductive alkylation. Several kinds of IgG against tag peptides and small molecules were successfully utilized to detect antigens in a rapid and quantitative manner.

Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis.

Membranes of living cells possess asymmetry. The inner and outer leaflets of the membrane consist of different phospholipid compositions, which are known to affect the function of membrane proteins, and the loss of the asymmetry has been reported to lead to cell apoptosis. In addition, different proteins are found on the inner and outer leaflets of the membrane, and they are essential for various biochemical reactions, including those related to signal transduction and cell morphology. While in vitro lipid bilayer reconstitution with asymmetric phospholipid compositions has been reported, the reconstitution of lipid bilayer where different proteins are localized in the inner and outer leaflet, thereby enables asymmetric protein localizations, has remained difficult. Herein, we developed a simple method to achieve this asymmetry using an in vitro transcription-translation system (IVTT). The method used a benzylguanine (BG) derivative-modified phospholipid, which forms a covalent bond with a snap-tag sequence. We show that purified snap-tagged protein can be localized to the cell-sized liposome surface via an interaction between BG and the snap-tag. We then show that IVTT-synthesized proteins can be located at the lipid membrane and that different proteins can be asymmetrically localized on the outer and inner leaflets of liposomes.

Statistical description of the denatured structure of a single protein, staphylococcal nuclease, by FRET analysis.

Structural characterization of fully unfolded proteins is essential for understanding not only protein-folding mechanisms, but also the structures of intrinsically disordered proteins. Because an unfolded protein can assume all possible conformations, statistical descriptions of its structure are most appropriate. For this purpose, we applied Förster resonance energy transfer (FRET) analysis to fully unfolded staphylococcal nuclease. Artificial amino acids labeled with a FRET donor or acceptor were introduced by an amber codon and a four-base codon respectively. Eight double-labeled proteins were prepared, purified, and subjected to FRET analysis in 6 M urea. The observed behavior could be explained by a power law, R = αN0.44, where R, and N are the distance and the number of residues between donor and acceptor, and α is a coefficient. The index was smaller than the value expected for an excluded-volume random coil, 0.588, indicating that the fully unfolded proteins were more compact than polypeptides in good solvent. The FRET efficiency in the native state did not necessarily correlate to the distance obtained from crystal structure, suggesting that other factors such as the orientation factor made a substantial contribution to FRET.

Freezing-Assisted Gene Delivery Combined with Polyampholyte Nanocarriers.

Physical methodologies such as electroporation and the gene-gun technology have been widely used for transfection; however, their applicability is limited because they lead to cell damage and low cell viability. Therefore, to address these limitations we developed a new freeze concentration-based gene transfection system that provides enhanced in vitro gene delivery compared to that provided by the commercially available systems. The system employs a facile freeze concentration step, whereby cells are simply frozen to very low temperatures in the presence of polymer-pDNA complexes. As part of system development, we also synthesized a low toxicity polyethylenimine (PEI)-based polyampholyte prepared through succinylation with butylsuccinic anhydride. In aqueous solution, this modified polyampholyte self-assembles to form small (20 nm diameter), positively charged (net surface charge of 35 mV), nanoparticles through a combination of hydrophobic and electrostatic interactions. Agarose gel electrophoresis analysis indicated that the polyampholyte nanoparticle was able to form a complex with pDNA that provided stability against nuclease degradation. Using transfection of HEK-293T cells, we demonstrated that a combination of polyampholyte: pDNA, at an appropriate ratio, and the freeze concentration method resulted in significant enhancement of GFP and luciferase expression compared to commercially available carriers. Endosomal escape of pDNA was also found to be increased when using the modified polyampholyte compared to branched PEI. This study suggests that the efficient combination of freeze concentration and the modified polyampholyte described here has great potential for in vitro gene therapy.

Novel biosensor system model based on fluorescence quenching by a fluorescent streptavidin and carbazole-labeled biotin.

In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole-labeled biotin. BODIPY-FL-aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position-specifically incorporated into Trp120 position of streptavidin by four-base codon method. On the other hand, carbazole-labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole-labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole-labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole-labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd.

Antibody-based fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine.

Fluorescent indicators for protein phosphorylation are very important in not only fundamental biology but also biomedical applications. In this study, we developed novel fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine (pTyr) derivatives. A single-chain antibody variable fragment (scFv) against phosphotyrosine was fluorescent-labeled by incorporation of tetramethylrhodamine (TAMRA)-linked nonnatural amino acid at the N- or C-terminus. The TAMRA-labeled scFv showed fluorescence enhancement upon addition of pTyr-containing peptides based on antigen-dependent fluorescence quenching effect on TAMRA. The TAMRA-labeled scFv was further fused with enhanced green fluorescent protein (EGFP) to generate a double-labeled scFv for pTyr. In the absence of antigen, fluorescence resonance energy transfer (FRET) occurred from EGFP to TAMRA but TAMRA was quenched. The antigen-binding removed the quenching of TAMRA while FRET occurred without altering its efficiency. As a result of the FRET and antigen-dependent fluorescence quenching effect, the double-labeled scFv exhibited fluorescence ratio enhancement upon the antigen-binding. The fluorescent and fluorescent ratiometric indicators obtained in this study will become a novel tool for analysis of protein phosphorylation. Moreover, this strategy utilizes antibody derivatives, and therefore, can be easily applied to other antigen-antibody pairs to generate fluorescent ratiometric indicators for various target molecules.

Incorporation of a Doubly Functionalized Synthetic Amino Acid into Proteins for Creating Chemical and Light-Induced Conjugates.

Z-Lysine (ZLys) is a lysine derivative with a benzyloxycarbonyl group linked to the ε-nitrogen. It has been genetically encoded with the UAG stop codon, using the pair of an engineered variant of pyrrolysyl-tRNA synthetase (PylRS) and tRNA(Pyl). In the present study, we designed a novel Z-lysine derivative (AmAzZLys), which is doubly functionalized with amino and azido substituents at the meta positions of the benzyl moiety, and demonstrated its applicability for creating protein conjugates. AmAzZLys was incorporated into proteins in Escherichia coli, by using the ZLys-specific PylRS variant. AmAzZLys was then site-specifically incorporated into a camelid single-domain antibody specific to the epidermal growth factor receptor (EGFR). A one-pot reaction demonstrated that the phenyl amine and azide were efficiently linked to the 5 kDa polyethylene glycol and a fluorescent probe, respectively, through specific bio-orthogonal chemistry. The antibody was then tested for the ability to form a photo-cross-link between its phenylazide moiety and the antigen, while the amino group on the same ring was used for chemical labeling. When incorporated at a selected position in the antibody and exposed to 365 nm light, AmAzZLys formed a covalent bond with the EGFR ectodomain, with the phenylamine moiety labeled fluorescently prior to the reaction. The present results illuminated the versatility of the ZLys scaffold, which can accommodate multiple reactive groups useful for protein conjugation.

Liposome-Based in Vitro Evolution of Aminoacyl-tRNA Synthetase for Enhanced Pyrrolysine Derivative Incorporation.

Methanosarcina species pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate amber suppressor tRNA. The introduction of two mutations (Y384F and Y306A) into PylRS was previously shown to generate a mutant, designated LysZ-RS, that was able to attach N-benzyloxycarbonyl-L-lysine (LysZ) to its cognate tRNA. Despite the potential of LysZ derivatives, further LysZ-RS engineering has not been performed; consequently, we aimed to generate LysZ-RS mutants with improved LysZ incorporation activity through in vitro directed evolution. Using a liposome-based in vitro compartmentalization (IVC) approach, we screened a randomly mutagenized gene library of LysZ-RS and obtained a mutant that showed increased LysZ incorporation activity both in vitro and in vivo. The ease and high flexibility of liposome-based IVC should enable the evolution of not only LysZ-RS that can attach various LysZ derivatives but also of other enzymes involved in protein translation.

A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins.

Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.

Incorporation of glycosylated amino acid into protein by an in vitro translation system.

O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.

Amber codon-mediated expanded saturation mutagenesis of proteins using a cell-free translation system.

Saturation mutagenesis of proteins, in which an amino acid at a specific site is substituted with each of the other 19 amino acids, is a powerful method for protein analysis and engineering. However, 19 mutated genes have to be prepared to express all possible amino acid-substituted proteins at one site. We previously reported a four-base codon-mediated saturation mutagenesis method for the expression of all 20 amino acid-substituted proteins from one four-base codon-containing gene using 20 types of chemically aminoacylated tRNAs corresponding to the four-base codon. In this study, an improved method for saturation mutagenesis using an amber codon was developed. By combining the use of Escherichia coli-derived amber suppressor tRNAs and chemically aminoacylated Mycoplasma-derived tRNAs, all 20 mutated proteins were successfully expressed from one amber mutant gene in a cell-free translation system. The use of E. coli-derived amber suppressor tRNAs simplified the preparation of the tRNA reagents required for saturation mutagenesis, and also improved the expression of some of the mutated proteins. The expressed mutant proteins were used to evaluate the effect of the amino acid substitutions on the ligand-binding activity. To further expand the possibilities of saturation mutagenesis, a series of nonnatural amino acids analogous to a naturally occurring amino acid was added to the amino acid repertoire. The expanded saturation mutagenesis was utilized to evaluate the effect of a series of atomic-level side chain substitutions on the protein activity.

Incorporation of unnatural non-α-amino acids into the N-terminus of proteins in a cell-free translation system.

Unnatural amino acid mutagenesis allows us to introduce unnatural α-amino acids into internal positions of proteins in response to expanded codons such as amber and four-base codons. To improve the unnatural amino acid mutagenesis, the incorporation of unnatural α-amino acids and non-α-amino acids into the N-terminus of proteins has been achieved using expanded initiation codons. Here, we describe the method for the incorporation of fluorescent-labeled non-α-amino acids into the N-terminus of proteins in a cell-free translation system.

"Quenchbodies": quench-based antibody probes that show antigen-dependent fluorescence.

Here, we describe a novel reagentless fluorescent biosensor strategy based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. Using a cell-free translation-mediated position-specific protein labeling system, we found that an antibody single chain variable region (scFv) that had been fluorolabeled at the N-terminal region showed a significant antigen-dependent fluorescence enhancement. Investigation of the enhancement mechanism by mutagenesis of the carboxytetramethylrhodamine (TAMRA)-labeled anti-osteocalcin scFv showed that antigen-dependency was dependent on semiconserved tryptophan residues near the V(H)/V(L) interface. This suggested that the binding of the antigen led to the interruption of a quenching effect caused by the proximity of tryptophan residues to the linker-tagged fluorophore. Using TAMRA-scFv, many targets including peptides, proteins, and haptens including morphine-related drugs could be quantified. Similar or higher sensitivities to those observed in competitive ELISA were obtained, even in human plasma. Because of its versatility, this "quenchbody" is expected to have a range of applications, from in vitro diagnostics, to imaging of various targets in situ.

Farnesyl pyrophosphate regulates adipocyte functions as an endogenous PPARγ agonist.

The cholesterol biosynthetic pathway produces not only sterols but also non-sterol mevalonate metabolites involved in isoprenoid synthesis. Mevalonate metabolites affect transcriptional and post-transcriptional events that in turn affect various biological processes including energy metabolism. In the present study, we examine whether mevalonate metabolites activate PPARγ (peroxisome-proliferator-activated receptor γ), a ligand-dependent transcription factor playing a central role in adipocyte differentiation. In the luciferase reporter assay using both GAL4 chimaera and full-length PPARγ systems, a mevalonate metabolite, FPP (farnesyl pyrophosphate), which is the precursor of almost all isoprenoids and is positioned at branch points leading to the synthesis of other longer-chain isoprenoids, activated PPARγ in a dose-dependent manner. FPP induced the in vitro binding of a co-activator, SRC-1 (steroid receptor co-activator-1), to GST (glutathione transferase)-PPARγ. Direct binding of FPP to PPARγ was also indicated by docking simulation studies. Moreover, the addition of FPP up-regulated the mRNA expression levels of PPARγ target genes during adipocyte differentiation induction. In the presence of lovastatin, an HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase inhibitor, both intracellular FPP levels and PPARγ-target gene expressions were decreased. In contrast, the increase in intracellular FPP level after the addition of zaragozic acid, a squalene synthase inhibitor, induced PPARγ-target gene expression. The addition of FPP and zaragozic acid promotes lipid accumulation during adipocyte differentiation. These findings indicated that FPP might function as an endogenous PPARγ agonist and regulate gene expression in adipocytes.

Biosynthesis of proteins containing modified lysines and fluorescent labels using non-natural amino acid mutagenesis.

The preparation of posttranslationally modified proteins is required to investigate the function and structure of modified proteins. However, homogeneously modified proteins are not easily isolated from natural sources or prepared using modification enzymes. Non-natural amino acid mutagenesis has enabled us to incorporate modified amino acids into specific positions of proteins in both cell-free and in-cell translation systems using tRNAs that are aminoacylated with modified amino acids. Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins. To introduce methyllysine, dimethyllysine, trimethyllysine, and acetyllysine, frameshift and amber suppressor tRNAs aminoacylated with modified lysines were synthesized by chemical aminoacylation and supplied to an Escherichia coli cell-free translation system. The immunodetection of the translation products indicated that the modified lysines were incorporated into streptavidin and histone H3 in a quantitative, position-specific manner. Calmodulin derivatives containing a fluorescent non-natural amino acid at the N-terminal region and modified lysines at the Lys115 position were also synthesized, and their binding activity to a calmodulin-binding peptide was analyzed by fluorescence correlation spectroscopy. The results obtained here demonstrate that this method is useful in preparing and analyzing naturally occurring and non-natural modified proteins.

Incorporation of fluorescent non-natural amino acids into N-terminal tag of proteins in cell-free translation and its dependence on position and neighboring codons.

Fluorescence labeling is a useful technique for structural and functional analyses of proteins. In a previous study, we developed position-specific incorporation of visible wavelength fluorescent non-natural amino acids carrying relatively small BODIPY fluorophores into proteins, in response to a four-base codon CGGG. Here, we have expanded this position-specific fluorescence labeling method to include relatively large non-natural amino acids carrying photostable rhodamine dyes. TAMRA-linked aminophenylalanine was synthesized and attached to a tRNA having a four-base anticodon, and its incorporation into proteins was examined in an Escherichia coli cell-free translation system. TAMRA-labeled amino acids were successfully incorporated into proteins, although incorporation was allowed only at the N-terminal region. Insertion of two codons encoding a stop codon in the +1 frame before four-base codon suppressed the expression of non-labeled proteins that may have been produced by spontaneous +1 frameshift upstream of the four-base codon. Alternation of the incorporation position affected the expression level of the TAMRA-labeled protein. In addition, alternation of upstream and downstream codons affected the efficiency and accuracy of TAMRA-labeled amino acid incorporation. Based on these results, a novel tag peptide was developed; it contained the four-base codon at the 9th position with optimized upstream and downstream codons. This tag peptide was effective for producing proteins with various fluorescent labels at the N-terminal region.

Screening of amber suppressor tRNAs suitable to introduce nonnatural amino acids into proteins by real-time monitoring of cell-free translation.

Incorporation of nonnatural amino acids into proteins is a useful technique to analyze protein structure and function. We have reported that amber suppressor tRNAs suitable for efficient and specific incorporation of nonnatural amino acids into proteins can be obtained by screening a wide variety of naturally occurring tRNAs in an E. coli. cell-free translation system. The amber suppressor activity of the tRNAs was evaluated by incorporation of a fluorescent nonnatural amino acid and fluorescent SDS-PAGE analysis of cell-free translation products, though the SDS-PAGE was troublesome and time-consuming. In this research, we developed an alternative method for the screening of amber suppressor tRNAs by real-time measurement of fluorescence resonance energy transfer (FRET) from GFP to BODIPY558-linked nonnatural amino acid, which was incorporated into the N-terminus of GFP by amber suppressor tRNAs. Using this method, we demonstrated that the screening of the amber suppressor activity of various prokaryotic Trp tRNAs was performed in a high-throughput manner.

Site-specific incorporation of PEGylated amino acids into proteins using nonnatural amino acid mutagenesis.

Site-directed incorporation of PEGylated nonnatural amino acids with 4, 8, and 12 repeated ethylene glycol units was examined in a cell-free translation system. PEGylated aminophenylalanine derivatives were successfully incorporated into proteins, whereas PEGylated lysines were not. The incorporation efficiency of the PEGylated amino acids decreased with an increase in PEG chain length. The present method will be useful for preparation of proteins which are PEGylated in a site-specific and quantitative manner.