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Biomedical subjects

Tomoaki Matsuura

Publications and source records attributed to Tomoaki Matsuura.

10 recordsLinked to original sources
PubMed2006

Nascent chain, mRNA, and ribosome complexes generated by a pure translation system.

Ribosome display is based on the concept that ternary complexes consisting of a nascent chain, ribosome, and mRNA can be generated, thereby establishing the linkage between genotype and phenotype that is essential for evolutionary experiments. With cell extract-based in vitro translation systems, it has been shown that ternary complexes can be generated by omitting the termination codon from the constructs, which can be stabilized at low temperature in the presence of high Mg2+ concentrations. Using an Escherichia coli-based reconstituted in vitro translation system (PURE system), in which all components necessary for the translation reaction were highly purified and reconstituted, ternary complexes could be generated equally well with a variety of sequences at the 3' end of the RNA, even those with a termination codon. Moreover, the generated complexes were stable at temperatures between 4 and 50 degrees C, and are thus highly stable in contrast to previous assumptions.

Cell-Free System↗
PubMed2006

Femtoliter compartment in liposomes for in vitro selection of proteins.

The aqueous compartment in liposomes provides a reaction resembling the cell and therefore is used as a microcompartment in which to study enzymatic reactions. However, regardless of their method of preparation, the heterogeneity in size of cell-size liposomes limits their potential uses. We established a strategy to estimate the internal aqueous volume of cell-size liposomes using a fluorescence-activated cell sorter (FACS). Reactions inside individual liposomes can be measured in a high-throughput format provided that the encapsulated proteins give rise to a fluorescent signal such as by exhibiting fluorescence themselves or by catalyzing production of a fluorescent compound. The strategy of volume estimation was applied to in vitro selection experiments. The green fluorescent protein (GFP) gene was encapsulated into liposomes together with an in vitro translation system. Here liposomes carrying a single copy of the gene were identified using the internal aqueous volume information of individual liposomes, and those exhibiting higher green fluorescence intensity were sorted by the FACS machine. This system was able to enrich those encoding GFP with higher fluorescence intensity over those with lower intensity. These results suggest the possibility of performing evolutionary experiments in an environment that mimics the cell.

Cell Compartmentation↗
PubMed2006

Functional Qbeta replicase genetically fusing essential subunits EF-Ts and EF-Tu with beta-subunit.

Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta, is a heterotetramer composed of a phage-encoded beta-subunit and three host-encoded proteins: the ribosomal protein S1 (alpha-subunit), EF-Tu, and EF-Ts. Several purification methods for Qbeta replicase were described previously. However, in our efforts to improve the production of Qbeta replicase, a substantial amount of the beta-subunit overproduced in Escherichia coli cells was found as insoluble aggregates. In this paper, we describe two kinds of method of producing Qbeta replicase. In one kind, both EF-Tu and EF-Ts subunits were expressed with the beta-subunit, and in the other kind, the beta-subunit was genetically fused with EF-Tu and EF-Ts. The fused protein, a single-chain alpha-less Qbeta replicase, was mostly found in the soluble fraction and could be readily purified. These results pave the way for the large-scale production of the highly purified form of this enzyme.

Enzyme Activation↗
PubMed2006

In vitro evolution of proteins.

Consecutive rounds of diversification and selection of the fittest is believed to be the main driving force for the evolution of life. For the evolution of life to proceed, all living cells are surrounded by a lipid bilayer that separates their own genes from the external environment and from those of other organisms. In this way, the genetic information of an individual is replicated on the basis of their phenotype; thus the enrichment of the fittest will occur. Hence, evolution is based on linkage between genotype and phenotype owing to the surrounding of the genetic material with a barrier. The linkage between genotype and phenotype is also known to be essential for the directed evolution of proteins. Indeed, systems for molecular evolution, including phage display, ribosome display, and in vitro compartmentalization, all satisfy this requirement in different ways. These systems have been shown to be powerful tools for high-throughput screening for the functions of proteins, screening as many as <10(12) molecules in 1 d. These selection systems in combination with various gene libraries yield proteins with improved or altered biophysical properties, and may even allow the generation of proteins with novel functions.

Amino Acid Sequence↗
PubMed2004

Strategies for selection from protein libraries composed of de novo designed secondary structure modules.

As more and more protein structures are determined, it has become clear that there is only a limited number of protein folds in nature. To explore whether the protein folds found in nature are the only solutions to the protein folding problem, or that a lack of evolutionary pressure causes the paucity of different protein folds found, we set out to construct protein libraries without any restriction on topology. We generated different libraries (all alpha-helix, all beta-strand and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand and beta-turn) in various proportions, based primarily on the patterning of polar and non-polar residues. From the analysis of proteins chosen randomly from the libraries, we found that a substantial portion of pure alpha-helical proteins show properties similar to native proteins. Using these libraries as a starting point, we aim to establish a selection system which allows us to enrich proteins with favorable folding properties (non-aggregating, compactly folded) from the libraries. We have developed such a method based on ribosome display. This selection is based on two concepts: (1) misfolded proteins are more sensitive to proteolysis, (2) misfolded and/or aggregated proteins are more hydrophobic. We show that by applying each of these selection criteria proteins that are compactly folded and soluble can be enriched over insoluble and random coil proteins.

Protein Folding↗
PubMed2003

Selection based on the folding properties of proteins with ribosome display.

Ribosome display is a powerful tool for selecting and evolving protein functions through ligand-binding. Here, this in vitro system was used to perform selection based on the folding properties of proteins, independent of specific ligand-binding. The selection is based on two properties of misfolded proteins: (1) increased sensitivity to proteolysis and (2) greater exposure of hydrophobic area. By targeting these properties, we show that compactly folded and soluble proteins can be enriched over insoluble and random coil proteins. This approach may be especially useful for selection and evolution of folded proteins from random sequence libraries.

Amino Acid Sequence↗
PubMed2002

Importance of compartment formation for a self-encoding system.

A self-encoding system designed to have strict "compartition" of the molecules, i.e., to contain only a single molecule of DNA in each compartment, was established, and its evolutionary fate was analyzed. The system comprised the Thermus thermophilus DNA polymerase gene as the informational molecule and its protein product replicating the gene as the functional molecule. Imposing strict compartition allows the self-encoding system to last up to at least the tenth generation, whereas the system ceased to work after the third generation when loose compartition initiated with 100 molecules was imposed. These results provide experimental evidence on the importance of compartition for the maintenance of a self-encoding system. In addition, the extent of diversity in self-replication activity of the compartments was found to be another vital difference in the evolutionary dynamics between the strict and loose compartitions. Although the system with strict compartition provides widely diversified activity of the compartments at each generation, the values of the activity diverge only within a small range in the system with loose compartition. When the variety in the activity of a compartment is small, functional selection becomes weak, and to conform Darwinian evolution may become unfeasible. Therefore, strict compartition is essential for the evolvability of a self-encoding system.

Amino Acid Substitution↗
PubMed2002

Construction and characterization of protein libraries composed of secondary structure modules.

Only a minute fraction of all possible protein sequences can exist in the genomes of all life forms. To explore whether physicochemical constraints or a lack of need causes the paucity of different protein folds, we set out to construct protein libraries without any restriction of topology. We generated different libraries (all alpha-helix, all beta-strand, and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand, and beta-turn) in various proportions, based primarily on the patterning of polar and nonpolar residues. We wished to explore that part of sequence space that is rich in secondary structure. The analysis of randomly chosen clones from each of the libraries showed that, despite the low sequence homology to known protein sequences, a substantial proportion of the library members containing alpha-helix modules were indeed helical, possess a defined oligomerization state, and showed cooperative chemical unfolding behavior. On the other hand, proteins composed of mainly beta-strand modules tended to form amyloid-like fibrils and were among the least soluble proteins ever reported. We found that a large fraction of members in non-beta-strand-containing protein libraries that are distant from natural proteins in sequence space possess unexpectedly favorable properties. These results reinforce the efficacy of applying binary patterning to design proteins with native-like properties despite lack of restriction in topology. Because of the intrinsic tendency of beta-strand modules to aggregate, their presence requires precise topologic arrangement to prevent fibril formation.

Amino Acid Sequence↗