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Biochem. Biophys. Res. Commun. 373, 48–52. Selection of mRNA 5´-untranslated region sequence with high translation efficiency through ribosome display. 2008

Mie, M., Shimizu, S., Takahashi, F. and Kobatake, E.

Notes: The authors developed an in vitro selection system that is based on ribosome display and favors identification of 5´-untranslated regions (UTRs) with high translation efficiencies. A 5´-UTR random library was created in which the 5´-UTRs were upstream of a polyhistidine-tag/Renilla luciferase-coding region. In vitro transcripts from this library were translated in vitro using the Flexi® Rabbit Reticulocyte Lysate System. The authors preferentially selected mRNAs with high translational efficiencies by shortening the translation time and capturing ternary complexes of mRNA, ribosome and nascent proteins. These complexes were captured using MagneHis™ Ni Particles. RNA was extracted from these complexes and used as a template in RT-PCR for the next round of selection. Before and after each round of selection, 9µl of RNA was translated in vitro, and 20µl of translated product was removed every 5 minutes to measure Renilla luciferase activity and monitor translation efficiency. Renilla luciferase was measured using the Renilla Luciferase Assay System. After two rounds of selection, RT-PCR products were cloned into a pUC18 vector, the sequences of the resulting plasmids were confirmed, and 0.5µg of plasmid was translated in vitro using the TNT® T7 Coupled Rabbit Reticulocyte Lysate System to further evaluate translation efficiency. (3963)

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Mol. Cell. Biol. 20(9), 2959-2969. Negative and translation termination-dependent positive control of FLI-1 protein synthesis by conserved overlapping 5' upstream open reading frames in Fli-1 mRNA. 2000

Sarrazin, S., Starck, J., Gonnet, C., Doubeikovski, A., Melet, F., Morle, F.

Notes: Authors used the Flexi® Rabbit Reticulocyte Lysate System to in vitro translate Fli-1 mRNA. The lysate was programmed with mRNAs containing mutated sequences to determine the mechanism of translation initiation of two different isoforms. (2210)

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J. Biol. Chem. 274, 7570-7575. Folding and assembly of type X collagen mutants that cause metaphyseal chondrodysplasia-type schmid. Evidence for co-assembly of the mutant and wild-type chains and binding to molecular chaperones. 1999

McLaughlin, S.H., Conn, S.N., Bulleid, N.J.

Notes: Transcribed cDNA for type X collagen using Promega's T7 RNA Polymerase. Translated this using Flexi® Rabbit Reticulocyte Lysate System in presence of semi-permeabilized HT1080 cells to allow for correct assembly of fibrillar collagens. (0714)

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J. Biol. Chem. 274, 14198-14203. GTP-dependent binding of ADP-riboxylation factor to coatomer in close proximity to the binding site for dilysine retrieval motifs and p23 1999

Zhao, L., Helms, J.B., Brunner, J., Wieland, F.T.

Notes: To produce site-specific photolabile ADP-ribosylation factor mutants, the amino acid of interest was mutated to the Amber stop codon in the cDNA. The cDNA was then in vitro transcribed and the resulting RNA translated in vitro with the Flexi® Rabbit Reticulocyte Lysate System supplemented with a suppressor tRNA charged in vitro with the L-4'-(3-trifluoromethyl-3H-diazirin-3-yl)-phenylalanine. The suppressor tRNA was used at 5µM in the reaction. The reaction was performed with labeled methionine to judge the success of the suppression via autoradiography. The photolabile protein was used to crosslink to proteins interacting with ADP-ribosylation factor. (0057)

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Proc. Natl. Acad. Sci. USA 96, 8516-8521. The cytosolic tail of class I MHC heavy chain is required for its dislocation by the human cytomegalovirus US2 and US11 gene products. 1999

Story, C.M., Furman, M.H., Ploegh, H.L.

Notes: Inserts in the pSP72 Vector were in vitro transcribed and translated using the Flexi® Rabbit Reticulocyte Lysate System. (0340)

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J. Immunol. 160, 2297-2307. Characterization of the interactions between MHC class I subunits: a systematic approach for the engineering of higher affinity variants of beta 2-microglobulin. 1998

Shields, M. J., Assefi, N., Hodgson, W., Kim, E. J., Ribaudo, R.K.

Notes: The authors co-translated mRNAs for beta 2 microglobulin and MHC class I proteins in Flexi® Rabbit Reticulocyte Lysate System with Canine Pancreatic Microsomal Membranes (CMMs). They generated mRNAs using RiboMAX™ T7 Large Scale RNA Production System. Treated transcription reactions with RQ1 RNase-Free DNase I after transcription to remove template DNA. (0393)

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J. Biol. Chem. 273, 22091-22095. The a1(VIII) and a2(VIII) chains of type VIII collagen can form stable homotrimeric molecules. 1998

Illidge, C., Kielty, C., Shuttleworth, A.

Notes: The Flexi® Rabbit Reticulocyte Lysate System was used for cell-free translation reaction in the presence of 4 x 105 digitonin-permeabilized HT-1080 fibroblasts. Two types VIII chains were expressed as well as the a1chain of type X collagen. The expressed proteins were assayed by protease digestion and thermal denaturation for their quaternary structure. The in vitro transcripts for the translation reaction were produced with the Riboprobe® System. (0995)

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Blood 90, 372-381. Calreticulin biosynthesis and processing in human myeloid cells: Demonstration of signal peptide cleavage and N-glycosylation. 1997

Denning, G.M., Leidal, K.G., Holst, V.A., Iyer, S.S., Pearson, D.W., Clark, J.R., Nauseef, W.M. and Clark, R.A.

Notes: PolyA+ RNA from HL-60 cells was in vitro translated using the Rabbit Reticulocyte Lysate System in the presence or absence of Canine Microsomal Membranes, then immunoprecipitated and analyzed by SDS-PAGE and fluorography. The calreticulin protein was presumably glycosylated by the membranes. The same results were obtained with in vitro transcripts of the calreticulin. (1617)

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EMBO J. 16, 2703-2716. Cell-free synthesis and assembly of connexins into functional gap junction membrane channels. 1997

Falk, M.M., Buehler, L.K., Kumar, N.M. and Gilula, N.B.

Notes: Several different gap junction channel subunit isotypes, also known as connexins, were synthesized in vitro in the presence of Canine Microsomal Membranes. Translation reactions were spun through a sucrose cushion to separate the membrane fraction from the supernatant. Connexins that integrated into the microsomes formed homo- and heterooligomeric structures. The assembled proteins in the microsome were fused with a synthetic membrane and single channel conductance was measured and found to be very similar to wild-type membranes. (1618)

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J. Neurosci. 17, 181-189. Conservation of topology, but not conformation, of the proteolipid proteins of the myelin sheath. 1997

Gow A., Gragerov A., Gard A., Colman D.R., Lazzarini R.A.

Notes: All cDNAs to be translated in vitro were subcloned into the pSP64 Poly(A) Vector. The vector was linearized and RNA transcribed from the SP6 promoter with the Riboprobe® in vitro Transcription System. The in vitro transcribed RNA was translated in the Rabbit Reticulocyte Lysate in the presence or absence of Canine Pancreatic Microsomal Membranes (CMMs). N-linked glycosylation sequences were engineered into the proteolipid protein to determine the orientation of the domains of the protein in the membrane. (1114)

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J. Neurosci. 17, 3379-3391. Frequency-dependent inactivation of mammalian A-type K+ channel KV1.4 regulated by Ca2+/calmodulin-dependent protein kinase. 1997

Roeper, J., Lorra, C., Pongs, O.

Notes: Two site-directed mutants of KV1.4 were expressed in the Flexi® Rabbit Reticulocyte Lysate System in the presence of Canine Pancreatic Microsomal Membranes (CMMs). The membranes were gently pelleted, resuspended and the proteins in vitro phosphorylated with Calmodulin-dependent protein kinase II. (0490)

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Cell 88, 757-766. Frxb, an execreted protein expressed in the Spemann Organizer, binds and inhibits Wnt-8. 1997

Wang. S., Krinks, M., Lin, K., Luyten, F.P. and Moos, M., Jr

Notes: To demonstrate an interaction between Frzb and Wnt proteins in vitro, the proteins were cotranslated using Rabbit Reticulocyte Lysate in the presence of Canine Microsomal Membranes then immunoprecipitated with specific antibodies to either one. Control immunoprecipitations were performed with cotranslations with Frzb and the β-lactamase control or Wnt and the β-lactamase control. The procedure for the immunoprecipitation is referenced. (1603)

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J. Cell Biol. 139, 895-905. Functional expression cloning and characterization of SFT, a stimulator of Fe transport. 1997

Gutierrez, J.A., Yu, J., Rivera, S., Wessling-Resnick, M.

Notes: The cRNA for the Stimulator of Fe Transport (SFT) was in vitro translated with Rabbit Reticulocyte Lysate in the presence of Canine Microsomal Membranes. The RRL was used at 70% of total volume and 2 equivalents of the membranes were used. After translation, the reaction was treated with RNase A to reduce background. The SFT protein is predicted to have six transmembrane domains. (1621)

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EMBO J. 16, 5807-5818. Interaction of MHC class II molecules with the invariant chain: Role of the invariant chain (81-90) region. 1997

Stumptner, P. and Benaroch, P.

Notes: Reactions were performed at 30°C in a 25µl volume with 3 equivalents of membranes. Following translation, the membranes were lysed with detergent and the proteins immunoprecipitated with various antisera. The immunoprecipitated proteins were also subjected to endoglycosidase H digestion. (1598)

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Proc. Natl. Acad. Sci. USA 94, 14066-14071. Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with sever N-terminal tranmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus. 1997

Meera, P., Wallner, M., Song, M. and Toro, L.

Notes: Different truncations of the protein of interest were in vitro translated in the presence of the membranes. The translation reaction were centrifuged and the pellet and supernatants assays for the presence of the protein product. (1628)

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J. Biol. Chem. 272, 23117-23122. Multiple dimeric forms of human CD69 result from differential addition of N-glycans to typical (Asn-X-Ser/Thr) and atypical (Asn-X-Cys) glycosylation motifs. 1997

Vance, B.A., Wu, W., Ribaudo, R.K., Segal, D.M. and Kearse, K.P.

Notes: The CD69 transcripts were prepared with the T3 RiboMAX® System and translated with the Flexi® System in the presence of the membranes. After 60 minutes at 30°C, the translation reaction was layered onto a sucrose solution and centrifuged at 100,000 x g for 20min. The supernatant was discarded and the pellet was resuspended in SDS-PAGE sample buffer or digested with endoglycosidase H. (1601)

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J. Neurosci. 17, 8201-8212. Neuronal alpha-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors. 1997

Rangwala, F., Drisdel, R.C., Rakhilin, S., Ko, E., Atluri, P., Harkins, A.B., Fox, A.P., Salman, S.B. and Green, W.N.

Notes: The two products were used to show that the rat a7 cDNA was translated and could be processed in vitro to a higher molecular weight, most likely by glycosylation. (1630)

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J. Biol. Chem. 272, 13750-13757. The N-terminal domain of human GABA Receptor rho 1 subunits contains signals for homooligomeric and heterooligomeric interaction. 1997

Hackan, A.S., Wang, T.L., Guggino, W.B. and Cutting, G.R.

Notes: The proteins r1 and r2 as well as the N-terminal half of r1 and the C-terminus of r1 were expressed using the Flexi® Rabbit Reticulocyte Lysate System in the presence of Canine Microsomal Membranes. The r1 protein has four membrane spanning domains. The orientation of the protein was determined by proteinase K digestions of intact membranes. Digestions were terminated by the addition of PMSF rather than direct lysis in SDS sample buffer. (1622)

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J. Biol. Chem. 272, 6119-6127. Topological rules for membrane protein assembly in eukaryotic cells. 1997

Gafvelin, G., Sakaguchi, M., Andersson, H. and von Heijne, G.

Notes: The paper looked at the insertion of a model protein with one, two and four transmembrane segments and different distributions of positively charged residues in the N-terminal tail and the polar loops. The proteins were translated in the presence of the microsomes. Translocation of polypeptides to the lumenal side of the microsomes was assayed by prevention of N-linked glycosylation through competitive inhibition by the addition of a glycosylation acceptor tripeptide but not by a nonacceptor tripeptide and by proteinase K treatment of the microsomes. All techniques are referenced. (2033)

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Proc. Natl. Acad. Sci. USA 94, 7633-7638. Transmembrane topology of a CLC chloride channel. 1997

Schmidt-Rose, T. and Jentsch, T.J.

Notes: Increasing residues of the chloride channel were fused to a prolactin epitope and translated in vitro using Promega's Rabbit Reticulocyte Lysate in the presence of Canine Microsomal Membranes. The proteins were subjected to a proteinase K digest in the presence or absence of Triton X-100. The protease digestions were stopped with PMSF and the translation reaction analyzed by Western blotting. The protease protection assays were used to define the topology of the channel. The chloride channel crosses the membrane at least 10 times. Very good protocol for the protease protection assay. (1596)

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J. Clin. Invest. 97, 910-914. Autoantibodies to the extracellular domain of the calcium sensing receptor in patients with acquired hypoparathyroidism. 1996

Li.Y., Song, Y.H., Rais, N., Connor, E., Schatz, D., Muir, A. and Maclaren, N.

Notes: The extracellular domain of the receptor was expressed in vitro using Promega's Rabbit Reticulocyte Lysate in the presence or absence of the microsomes. The 60kDa extracellular domain is processed to a 70kDa glycoprotein in the presence of the membranes. The paper also shows a titration of the membranes in the translation reaction from 1-4µl per reaction. More protein is processed with increasing amounts of the microsomes. (1627)

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Proc. Natl. Acad. Sci. USA 93, 14922-14927. Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca2+-sensitive K+ channels: An additional transmembrane region at the N terminus. 1996

Wallner, M., Meera, P. and Toro, L.

Notes: Truncation mutants of the protein of interest were translated in the presence of the membranes. The membranes were pelleted and analyzed directly or treated with endoglycosidase F. The supernatents from the centrifugation were acetone precipitated and analyzed as well. (1602)

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J. Biol. Chem. 271, 26810-26818. Determinants of membrane association for poliovirus protein 3AB. 1996

Towner, J.S., Ho, T.V. and Semler, B.L.

Notes: The 3AB protein was fused with a FLAG tag. The rabbit lysate was centrifuged prior to use to remove any protein aggregates. The reaction was set up in a 200µl volume with the 3AB protein transcript and a b globin mRNA. The reaction was split into two 100µl reactions and to one 25 equivalents of membranes were added and to the other, just buffer. The translation reactions were allowed to procede for 30min at 30°C, then translation stopped by the addition of cycloheximide to 300µM. The reactions were centrifuged at through a sucrose gradient at 86,000 x g. Fractions were removed and subjected to immunoprecipitation and gel analysis. The 3AB protein was found to be membrane associated. Further studies with the addition of the Canine Microsomal Membranes after translation and cycloheximide demonstrated a membrane association after translation. (1599)

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J. Biol. Chem. 271, 25506-25514. Formation of a ligand-binding site for the acetylcholine receptor in vitro. 1996

Shtrom, S.S. and Hall, Z.W.

Notes: Translations with the Flexi Rabbit Reticulocyte Lysate System were performed in 25µl volume containing 3 equivalents of membranes. Reactions were performed at 28.5°C and in the presence or absence of the glycosylation inhibitor AcNYT. Translation products were analyzed either directly by SDS-PAGE followed by fluorography or with prior immunoprecipitation of the translation products. Capped transcripts were produced by in vitro transcription with SP6 RNA Polymerase. (2029)

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J. Clin. Invest. 98, 2580-2587. Molecular cloning and characterization of the vasopressin-regulated urea transporter of rat kidney collecting ducts. 1996

Shayakul, C., Steel, A. and Hediger, M.A.

Notes: The novel UT1 urea transporter was translated in vitro in the presence or absence of the microsomes. In the absence of the microsomes, the protein migrates at 76kDa and in the presence, 86kDa. The data is not shown but described in the text. (1597)

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