Femtoliter compartment in liposomes for in vitro selection of proteins

Sunami, Takeshi; Sato, Kazunao; Matsuura, Tomoaki; Tsukada, Koji; Urabe, Itaru; Yomo, Tetsuya

doi:10.1016/j.ab.2006.06.040

Cited by 99 publications

(109 citation statements)

References 33 publications

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“…S2B). The enrichment factor was higher with smaller GUVs, as expected (19,20). This trend occurs because larger GUVs tend to encapsulate multiple genes, thus resulting in a reduction of the enrichment factor.…”

Section: Significancementioning

confidence: 49%

In vitro evolution of α-hemolysin using a liposome display

Fujii

Matsuura

Sunami

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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131

139

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Significance The directed evolution of proteins in vitro has generated highly functional proteins and has contributed to elucidating the sequence–function relationship of proteins. However, the available methods consider globular proteins, not membrane proteins, despite the biological and pharmaceutical importance of the latter. We report the development of a method called liposome display, which enables the in vitro evolution of membrane proteins. We applied the method to evolve the pore-forming activity of α-hemolysin from Staphylococcus aureus and obtained a mutant with 30-fold higher activity than the WT. Given its high degree of controllability, liposome display allows for the rapid and efficient evolution of a wide range of membrane proteins, thereby improving the field of membrane protein engineering.

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Section: Significancementioning

confidence: 49%

In vitro evolution of α-hemolysin using a liposome display

Fujii

Matsuura

Sunami

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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131

139

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“…Although we are still at the starting point of investigating the precise roles of the modifier proteins on the reaction, combining the experimental data with the PPI network may provide insight into how these proteins interact with the translation machinery. Moreover our data may be useful not only for technologies utilizing in vitro translation systems (19,48,49) but also for designing in vitro translation systems with significantly improved performance than those available at present and also for protein production in vivo, such as by coexpressing beneficial components together with a protein of interest.…”

Section: Discussionmentioning

confidence: 99%

Comprehensive Analysis of the Effects of Escherichia coli ORFs on Protein Translation Reaction

Kazuta¹,

Adachi²,

Matsuura³

et al. 2008

Molecular & Cellular Proteomics

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Protein synthesis is one of the most important reactions in the cell. Recent experimental studies indicated that this complex reaction can be achieved with a minimum complement of 36 proteins and ribosomes by reconstituting an Escherichia coli-based in vitro translation system with these protein components highly purified on an individual basis. From the protein-protein interaction (PPI) network of E. coli proteins, these minimal protein components are known to interact physically with large numbers of proteins. However, it is unclear what fraction of E. coli proteins are linked functionally with the minimal protein synthesis system. We investigated the effects of each of the 4194 E. coli ORF products on the minimal protein synthesis system; at least 12% of the entire ORF products, a significant fraction of the gene product of E. coli, affect the activity of this system. Furthermore 34% of these functional modifiers present in the PPI network were shown by mapping to be directly linked (i.e. to interact physically) with the minimal components of the PPI network. Topological analysis of the relationships between modifiers and the minimal components in the PPI network indicated clustering of the minimal components. The modifiers showed no such clustering, indicating that the location of functional modifiers is spread across the PPI network rather than clustering close to the minimal protein components. These observations may reflect the evolutionary process of the protein synthesis system. Molecular & Cellular Proteomics 7:1530 -1540, 2008.The protein translation reaction (1), one of the most important regulators of cell behavior, involves the interactions of a large number of components and can thus be seen as an intermolecular interaction network. It has been demonstrated experimentally that 36 enzymes and the ribosomes are sufficient to carry out protein translation (2). These minimal protein components include the ribosomal proteins; initiation, elongation, and release factors; aminoacyl-tRNA synthetases; and enzymes involved in energy regeneration. This was demonstrated by constructing an Escherichia coli-based reconstituted in vitro translation system with these protein components highly purified on an individual basis.Although the genome of E. coli contains more than 4000 genes (3), constituting a very large interaction network (4, 5), the number of protein components constituting the minimal protein synthesis system corresponds to only 2.1% of the genes encoded in the genome. Thus, only small subsets of the protein components are required for protein synthesis. On the other hand, a number of previous studies, including protein-protein interaction (PPI) 1 network analysis in E. coli (4, 5), indicated that protein components constituting the minimal protein synthesis system interact with a large number of other proteins. To gain a deeper understanding of the protein translation system, it is important to identify not only the proteins that interact physically but also those that interact functionally, i.e. t...

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“…Starting from 2006, the use of PURE system for synthesizing a protein inside liposomes has become the benchmark for SSMCs construction (Sunami et al, 2006;Murtas et al, 2007), even if the yield of produced protein is typically one third when compared with cell extracts (Hillebrecht & Chong, 2008). Interestingly, we can look at the PURE system as a synthetic biology tool from the viewpoint of "standard biological parts" (http://partsregistry.org/Main_Page).…”

Section: The Technology For Building Minimal Cellsmentioning

confidence: 99%

Advances in Minimal Cell Models: a New Approach to Synthetic Biology and Origin of Life

Stano¹

2011

Progress in Molecular and Environmental Bioengineering - From Analysis and Modeling to Technology Applications

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Femtoliter compartment in liposomes for in vitro selection of proteins

Cited by 99 publications

References 33 publications

In vitro evolution of α-hemolysin using a liposome display

In vitro evolution of α-hemolysin using a liposome display

Comprehensive Analysis of the Effects of Escherichia coli ORFs on Protein Translation Reaction

Advances in Minimal Cell Models: a New Approach to Synthetic Biology and Origin of Life

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