Cell-free protein synthesis: applications in proteomics and biotechnology

He, Mingyue

doi:10.1016/j.nbt.2008.08.004

Cited by 74 publications

(56 citation statements)

References 73 publications

(101 reference statements)

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“…Transcription/translation extracts are the main systems to carry out gene expression in vitro. Originally prepared to study biological processes in vitro (21), modern cell-free systems have been developed for large-scale protein synthesis in response to an increasing number of applications in biotechnology (22). A cytoplasmic extract, prepared from a living organism, provides the translation machinery.…”

Section: Bottom-up Development Of An Artificial Cell: Broad Consideramentioning

confidence: 99%

Development of an artificial cell, from self-organization to computation and self-reproduction

Noireaux¹,

Maeda

Libchaber

2011

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

295

267

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This article describes the state and the development of an artificial cell project. We discuss the experimental constraints to synthesize the most elementary cell-sized compartment that can self-reproduce using synthetic genetic information. The original idea was to program a phospholipid vesicle with DNA. Based on this idea, it was shown that in vitro gene expression could be carried out inside cell-sized synthetic vesicles. It was also shown that a couple of genes could be expressed for a few days inside the vesicles once the exchanges of nutrients with the outside environment were adequately introduced. The development of a cell-free transcription/translation toolbox allows the expression of a large number of genes with multiple transcription factors. As a result, the development of a synthetic DNA program is becoming one of the main hurdles. We discuss the various possibilities to enrich and to replicate this program. Defining a program for self-reproduction remains a difficult question as nongenetic processes, such as molecular self-organization, play an essential and complementary role. The synthesis of a stable compartment with an active interface, one of the critical bottlenecks in the synthesis of artificial cell, depends on the properties of phospholipid membranes. The problem of a self-replicating artificial cell is a long-lasting goal that might imply evolution experiments.Defining Life Since Schrödinger's classic book What Is Life? (1), the definition of life has always been a puzzle with a variety of partial answers, none really satisfying. One answer is that life is a coded system with mutation and error correction (2). The information is encoded into DNA (the genotype) and the genetic code allows translating this information into proteins (the phenotype). The genetic code is universal, and the genome can support mutations, recombination, duplication, and partial transfer from organisms to organisms. Error correction limits the risk of melting the information into random sequences. This is fine, but life is also metabolism with a permanent absorption and transformation of nutrients from the environment (3). A constant flux of energy is needed to sustain the operation of this living dynamical system out of equilibrium. But life is also self-reproduction (4). Cells originate from preexisting cells by cell division. The DNA genome is replicated, the cell self-reproduces as well as the complete organism. Is self-reproduction a constraint of evolution present at each step and imposing severe design constraints or a possible definition of life? Or is life an emerging phenomenon resulting from complex chemical reactions, developing networks and cycles until, at a certain critical size of this network, life starts as an emerging property (5)? Within this approach, life is a dynamical system where signal to noise is just marginal; a living system positions itself at the edge of chaos. Finally the only generally accepted theme is that life is the result of evolution, natural selection, and adaptation (6), a...

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Section: Bottom-up Development Of An Artificial Cell: Broad Consideramentioning

confidence: 99%

Development of an artificial cell, from self-organization to computation and self-reproduction

Noireaux¹,

Maeda

Libchaber

2011

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

295

267

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“…Rather commonly, the eukaryotic proteins and their domains are expressed in the Escherichia coli bacterial cells (1)(2)(3) or cell-free extracts (3)(4)(5)(6). However, only a minor fraction of all heterologous proteins can be successively expressed in this host system.…”

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confidence: 99%

Multiple Post-translational Modifications Affect Heterologous Protein Synthesis

Tokmakov

Kurotani

Takagi

et al. 2012

Journal of Biological Chemistry

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Background: Post-translational modifications (PTMs) affect protein folding. Results: Statistically significant correlations are revealed between the yield of heterologous protein expression and the presence of multiple PTM sites bioinformatically predicted in the expressed sequences. Conclusion: Predicting potential PTMs in polypeptide sequences can help optimize heterologous protein synthesis. Significance: Correlations revealed provide insights into the role of specific PTMs in protein stability and solubility.

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“…An array of 3 × 4 wells was created in the footprint of a 96 well plate; for simplicity (and less waste), we did not make a 12 × 8 array. In the top layer, the largest well is a 4.5 mm diameter reaction chamber with a volume of 79.5 mm 3 , and it is surrounded by three 2 mm diameter access holes connecting to the feeding chambers. The feeding chambers on the bottom layer are 12 units of 9 mm diameter wells with a depth of 3 mm and a volume of 188.6 mm 3 ; each unit encompasses both reaction chamber and access holes in the top layer.…”

Section: Device Fabricationmentioning

confidence: 99%