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Etsuko Miyamoto-Sato

Publications and source records attributed to Etsuko Miyamoto-Sato.

11 recordsLinked to original sources
PubMed2006

In vitro evolution of single-chain antibodies using mRNA display.

Here we describe the application of the in vitro virus mRNA display method, which involves covalent linkage of an in vitro-synthesized antibody (phenotype) to its encoding mRNA (genotype) through puromycin, for in vitro evolution of single-chain Fv (scFv) antibody fragments. To establish the validity of this approach to directed antibody evolution, we used random mutagenesis by error-prone DNA shuffling and off-rate selection to improve the affinity of an anti-fluorescein scFv as a model system. After four rounds of selection of the library of mRNA-displayed scFv mutants, we obtained six different sequences encoding affinity-matured mutants with five consensus mutations. Kinetic analysis of the mutant scFvs revealed that the off-rates have been decreased by more than one order of magnitude and the dissociation constants were improved approximately 30-fold. The antigen-specificity was not improved by affinity maturation, but remained similar to that of the wild type. Although the five consensus mutations of the high-affinity mutants were scattered over the scFv sequence, analysis by site-directed mutagenesis demonstrated that the critical mutations for improving affinity were the two that lay within the complementarity determining regions (CDRs). Thus, mRNA display is expected to be useful for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.

Antibody Affinity↗
PubMed2006

Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy.

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein-protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca2+-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein-protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.

Calmodulin↗
PubMed2006

Affinity selection of DNA-binding protein complexes using mRNA display.

Comprehensive analysis of DNA-protein interactions is important for mapping transcriptional regulatory networks on a genome-wide level. Here we present a new application of mRNA display for in vitro selection of DNA-binding protein heterodimeric complexes. Under improved selection conditions using a TPA-responsive element (TRE) as a bait DNA, known interactors c-fos and c-jun were simultaneously enriched about 100-fold from a model library (a 1:1:20 000 mixture of c-fos, c-jun and gst genes) after one round of selection. Furthermore, almost all kinds of the AP-1 family genes including c-jun, c-fos, junD, junB, atf2 and b-atf were successfully selected from an mRNA display library constructed from a mouse brain poly A(+) RNA after six rounds of selection. These results indicate that the mRNA display selection system can identify a variety of DNA-binding protein complexes in a single experiment. Since almost all transcription factors form heterooligomeric complexes to bind with their target DNA, this method should be most useful to search for DNA-binding transcription factor complexes.

Animals↗
PubMed2006

Toward functional analysis of protein interactome using "in vitro virus": in silico analyses of Fos/Jun interactors.

Our high-throughput in vitro virus (IVV) method for selection of protein-protein interactions (PPI) and complexes, based on a simple cell-free co-translation and selection followed by computational sequence data analysis, was previously used to identify 31 Fos and Jun interactors. Here, in silico analyses of biological function, localization and phenotype of these AP-1 (Fos/Jun) interactors were performed. The results suggest that Fos and Jun do not necessarily work together, but also interact separately with novel interactors, including products of disease-related genes. Fos showed transcription-related activities, while Jun interacted with motor-related and structural proteins. The reliability of the IVV selection for the Fos interactors was further confirmed by means of in vitro reciprocal prey and bait protein experiments and co-immunoprecipitation. Further study of these novel interactors may provide clues to new pathways or mechanisms of biological functions and diseases.

Animals↗
PubMed2005

Subdivision of large introns in Drosophila by recursive splicing at nonexonic elements.

Many genes with important roles in development and disease contain exceptionally long introns, but special mechanisms for their expression have not been investigated. We present bioinformatic, phylogenetic, and experimental evidence in Drosophila for a mechanism that subdivides many large introns by recursive splicing at nonexonic elements and alternative exons. Recursive splice sites predicted with highly stringent criteria are found at much higher frequency than expected in the sense strands of introns >20 kb, but they are found only at the expected frequency on the antisense strands, and they are underrepresented within introns <10 kb. The predicted sites in long introns are highly conserved between Drosophila melanogaster and Drosophila pseudoobscura, despite extensive divergence of other sequences within the same introns. These patterns of enrichment and conservation indicate that recursive splice sites are advantageous in the context of long introns. Experimental analyses of in vivo processing intermediates and lariat products from four large introns in the unrelated genes kuzbanian, outspread, and Ultrabithorax confirmed that these introns are removed by a series of recursive splicing steps using the predicted nonexonic sites. Mutation of nonexonic site RP3 within Ultrabithorax also confirmed that recursive splicing is the predominant processing pathway even with a shortened version of the intron. We discuss currently known and potential roles for recursive splicing.

Alternative Splicing↗
PubMed2005

Cell-free cotranslation and selection using in vitro virus for high-throughput analysis of protein-protein interactions and complexes.

We have developed a simple and totally in vitro selection procedure based on cell-free cotranslation using a highly stable and efficient in vitro virus (IVV). Cell-free cotranslation of tagged bait and prey proteins is advantageous for the formation of protein complexes and allows high-throughput analysis of protein-protein interactions (PPI) as a result of providing in vitro instead of in vivo preparation of bait proteins. The use of plural selection rounds and a two-step purification of the IVV selection, followed by in vitro post-selection, is advantageous for decreasing false positives. In a single experiment using bait Fos, more than 10 interactors, including not only direct, but also indirect interactions, were enriched. Further, previously unidentified proteins containing novel leucine zipper (L-ZIP) motifs with minimal binding sites identified by sequence alignment as functional elements were detected as a result of using a randomly primed cDNA library. Thus, we consider that this simple IVV selection system based on cell-free cotranslation could be applicable to high-throughput and comprehensive analysis of PPI and complexes in large-scale settings involving parallel bait proteins.

Animals↗
PubMed2004

In vitro selection of Jun-associated proteins using mRNA display.

Although yeast two-hybrid assay and biochemical methods combined with mass spectrometry have been successfully employed for the analyses of protein-protein interactions in the field of proteomics, these methods encounter various difficulties arising from the usage of living cells, including inability to analyze toxic proteins and restriction of testable interaction conditions. Totally in vitro display technologies such as ribosome display and mRNA display are expected to circumvent these difficulties. In this study, we applied an mRNA display technique to screening for interactions of a basic leucine zipper domain of Jun protein in a mouse brain cDNA library. By performing iterative affinity selection and sequence analyses, we selected 16 novel Jun-associated protein candidates in addition to four known interactors. By means of real-time PCR and pull-down assay, 10 of the 16 newly discovered candidates were confirmed to be direct interactors with Jun in vitro. Furthermore, interaction of 6 of the 10 proteins with Jun was observed in cultured cells by means of co-immunoprecipitation and observation of subcellular localization. These results demonstrate that this in vitro display technology is effective for the discovery of novel protein-protein interactions and can contribute to the comprehensive mapping of protein-protein interactions.

Animals↗
PubMed2003

Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins.

For high-throughput in vitro protein selection using genotype (mRNA)-phenotype (protein) fusion formation and C-terminal protein labeling as a post-selection analysis, it is important to improve the stability and efficiency of mRNA templates for both technologies. Here we describe an efficient single-strand ligation (90% of the input mRNAs) using a fluorescein-conjugated polyethylene glycol puromycin (Fluor-PEG Puro) spacer. This ligation provides a stable c-jun mRNA with a flexible Fluor-PEG Puro spacer for efficient fusion formation (70% of the input mRNA with the PEG spacer) in a cell-free wheat germ translation system. When using a 5' untranslated region including SP6 promoter and Omega29 enhancer (a part of tobacco mosaic virus Omega), an A(8) sequence (eight consecutive adenylate residues) at the 3' end is suitable for fusion formation, while an XA(8) sequence (XhoI and the A(8) sequence) is suitable for C-terminal protein labeling. Further, we report that Fluor-PEG N-t-butyloxycarbonylpuromycin [Puro(Boc)] spacer enhances the stability and efficiency of c-jun mRNA template for C-terminal protein labeling. These mRNA templates should be useful for puromycin-based technologies (fusion formation and C-terminal protein labeling) to facilitate high-throughput in vitro protein selection for not only evolutionary protein engineering, but also proteome exploration.

3' Untranslated Regions↗
PubMed2003

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins.

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.

Animals↗
PubMed2002

Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions.

We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.

DNA-Binding Proteins↗