() Schematic illustration of the formation of streptavidin–tRNA fusion using puromycin–tRNA, which contains a puromycin moiety in the place of 3′ terminal aminoacyl-adenosine and a four-base anticodon CCCG

<p><b>Copyright information:</b></p><p>Taken from " selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an cell-free translation system"</p><p>Nucleic Acids Research 2006;34(5):e44-e44.</p><p>Published online 20 Mar 2006</p><p>PMCID:PMC1405820.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The puromycin–tRNA binds to ribosomal A site and accepts a streptavidin polypeptide chain as an analog of aminoacyl-tRNA in response to a four-base CGGG codon at 3′ terminus of streptavidin mRNA in a cell-free translation. The resulting streptavidin–puromycin–tRNA may be translocated to the P-site. In this case, the next aminoacyl-tRNA binds to the vacant ribosomal A site, but can not accept the polypeptide chain because of the amide bond of puromycin–tRNA. The resulting streptavidin–tRNA fusion is released from the ribosome complex by the addition of EDTA. () Schematic illustration of the selection system of tRNAs. Step 1, a DNA pool encoding tRNAs containing a four-base anticodon CCCG is transcribed by T7 RNA polymerase to tRNA(-CA) pool. Step 2, the tRNA(-CA) pool is ligated with pdCp-Puromycin by T4 RNA ligase to generate puromycin–tRNA. Step 3, a streptavidin mRNA containing a four-base CGGG codon at C-terminus is translated in an cell-free translation system in the presence of the puromycin–tRNA. Puromycin–tRNAs that successfully decode the CGGG codon form ribosome–mRNA–streptavidin–tRNA complex. Step 4, the streptavidin–tRNA fusion is dissociated from the complex by the addition of EDTA. Step 5, the streptavidin–tRNA fusion is recovered with biotin-coated magnetic beads. Step 6, the streptavidin–tRNA fusion is dissociated from the beads, and then the tRNA moiety is subjected to RT–PCR. Step 7, the tRNA genes are regenerated by overlap-extension PCR with a T7 promoter primer, which are used as template DNAs in the next round of selection

Formation of streptavidin–tRNA fusion and recovery of the corresponding tRNA gene using yeast

<p><b>Copyright information:</b></p><p>Taken from " selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an cell-free translation system"</p><p>Nucleic Acids Research 2006;34(5):e44-e44.</p><p>Published online 20 Mar 2006</p><p>PMCID:PMC1405820.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () An mRNA sequence used for the formation of streptavidin–tRNA fusion. The streptavidin gene contains T7 tag at N-terminus for the detection by western blot analysis, and five CGGG codons at C-terminus through a linker sequence consisted of GlyGlySerGlyGlySer sequence. () Western blot analysis of the formation of streptavidin–tRNA fusion by adding the streptavidin mRNA to a cell-free translation system in the absence of the puromycin–tRNA, in the presence of the puromycin–tRNA, and in the presence of the puromycin-tRNA and after treatment with RNaseA. () The amount of tRNA recovered with biotin-coated magnetic beads as streptavidin–tRNA fusion was determined by quantitative PCR analysis. The tRNAs were recovered from the cell-free translation products obtained in the presence of mRNA without CGGG codon, and with five CGGG codons at C-terminus. () PAGE analysis of the RT–PCR product of the recovered tRNAs obtained in the presence of mRNA without CGGG codon, and with five CGGG codons at C-terminus. The RT–PCR product of the yeast (-CA) was applied as a positive control

() Western blot analysis of the expression of full-length streptavidin in the presence of non-aminoacylated tRNAs to examine orthogonality of the tRNAs against endogenous aminoacyl-tRNA synthetases

<p><b>Copyright information:</b></p><p>Taken from " selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an cell-free translation system"</p><p>Nucleic Acids Research 2006;34(5):e44-e44.</p><p>Published online 20 Mar 2006</p><p>PMCID:PMC1405820.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Relative yield of the full-length streptavidin. The data were represented as means ± SD of three assays

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

A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins

<div><p>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.</p></div

List of constructs used.

<p>List of constructs used.</p

Position-specific incorporation of fluorescent amino acids by the cell-free system.

<p>RNA templates encoding variants of KvAP, pro-TNF, or Syb2 were translated in the cell-free system supplemented with BODIPYFL-AF conjugated tRNA with or without the addition of Sf21microsomes (microsomes). The non-natural amino acids were incorporated into introduced TAG codon. The resulting proteins were subjected to SDS-PAGE (10-15% polyacrylamide) and detected with excitation at 488 nm and emission at 530 nm. The amber codon in the different constructs was substituted for the codon at the position corresponding to the listed amino acids (except for M6 in pro-TNF where the TAG codon was inserted as an additional codon after the M6 codon) and marked by a star in the diagram.</p

Reconstitution of the AspT aspartate transporter synthesized in the cell-free system with Sf21 microsomes.

<p>(A) Cell-free synthesized AspT purified with cobalt affinity resin under non-denaturing conditions was solubilized with 1.5% DDM, and eluted with 0.01% DDM and 250 mM imidazole. The purified AspT protein (arrow head) was subjected to SDS-PAGE. (B) L-aspartate transport activity of proteoliposomes containing purified AspT protein. Proteoliposomes loaded with 100 mM L-aspartate were resuspended in 50 mM phosphate buffer (pH 7) (8.3 µg protein/ml). At 0 min, L-[³H] aspartate was added into the buffer (2.5 mM final concentration). After the rate of influx and efflux of L-[³H] aspartate was equal (at steady state), non-radiolabelled L-aspartate was added to a final concentration of 15 mM at 7.5 min (solid line, arrow indicating time of addition of unlabelled substrate). The broken line corresponds to the same experiment performed without addition of unlabelled L-aspartate. (C) Comparison of the initial uptake rates for L-aspartate into <i>E. coli</i> expressing AspT (hatched bar) and into microsomes containing cell-free synthesized AspT (solid bar). The transport activity at 1 min was regarded as the initial uptake rate.</p

Production of different membrane proteins using the cell-free system.

<p>After synthesis of the membrane proteins in the cell-free system in the presence of FluoroTect Green_Lys tRNA, the membrane fraction was precipitated and separated by SDS/PAGE. (A) 10% polyacrylamide gel; lane 1, AtKC1 (plant, K⁺ channel, 6 transmembrane segments (TMS)); lane 2, KAT1 (plant, K⁺ channel, 6 TMS); lane 3, KAT2 (plant, K⁺ channel, 6 TMS); lane 4, GORK (plant, K⁺ channel, 6 TMS); lane 5, AKT1 (plant, K⁺ channel, 6 TMS); lane 6, NhaA (bacteria, Na⁺/H⁺ antiporter, 10 TMS); Lane 7, AspT (bacteria, aspartate: alanine antiporter, 10 TMS); lane 8, MsbA (bacteria, ABC transporter, 12 TMS). (B) 15% polyacrylamide gel; lane 9, Sec61 β(human, 1 TMS).</p

Incorporation of fluorescent non-natural amino acids into NhaA.

<p>(A) Five version of NhaA (1–5) with introduced amber (TAG) codons (or Gly-Gly-TAG, introduced changes in bold) that enable incorporation of fluorescent non-natural amino acids into the N-terminal, middle or C-terminal regions of the protein (top panel) were expressed in the insect cell-free translation and translocation system in the presence of different fluorescent non-natural amino acids. The valine (V388) at the C-terminal end of NhaA was fused to additional sequences in the linker of the vectors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112874#pone-0112874-t001" target="_blank">Table 1</a>). Labeled NhaA protein was subjected to SDS-PAGE and fluorescent images were taken at the indicated excitation and emission wavelengths (bottom panel). The structure of the different fluorescent non-natural amino acids used in the experiments is shown above the gel images. (B) Incorporation of fluorescent non-natural amino acids into N-terminal or C-terminal positions in NhaA at an introduced four-base codon (CGGG) (top panel). NhaA was synthesized using the cell-free translation and translocation system in the presence of BODIPYFL-AF, subjected to SDS-PAGE and fluorescent images taken at the indicated excitation and emission wavelengths (bottom panel). (C) Double-labeling of NhaA by incorporation of BODIPY558-AF at an amber codon introduced into the N-terminal region and BODIPYFL-AF at the CGGG four-base codon introduced into the C-terminal region (top panel). NhaA was synthesized using the cell-free translation and translocation system in the presence of one or both fluorescent amino acids. Images of the SDS-PAGE were taken at the two different excitation and emission wavelengths indicated (bottom panel).</p