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Towards synthetic cells using peptide-based reaction compartments.

ABSTRACT: Membrane compartmentalization and growth are central aspects of living cells, and are thus encoded in every cell's genome. For the creation of artificial cellular systems, genetic information and production of membrane building blocks will need to be coupled in a similar manner. However, natural biochemical reaction networks and membrane building blocks are notoriously difficult to implement in vitro. Here, we utilized amphiphilic elastin-like peptides (ELP) to create self-assembled vesicular structures of about 200 nm diameter. In order to genetically encode the growth of these vesicles, we encapsulate a cell-free transcription-translation system together with the DNA template inside the peptide vesicles. We show in vesiculo production of a functioning fluorescent RNA aptamer and a fluorescent protein. Furthermore, we implement in situ expression of the membrane peptide itself and finally demonstrate autonomous vesicle growth due to the incorporation of this ELP into the membrane.

SUBMITTER: Vogele K

PROVIDER: S-EPMC6154970 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Towards synthetic cells using peptide-based reaction compartments.

Vogele Kilian K Frank Thomas T Gasser Lukas L Goetzfried Marisa A MA Hackl Mathias W MW Sieber Stephan A SA Simmel Friedrich C FC Pirzer Tobias T

Nature communications 20180921 1

Membrane compartmentalization and growth are central aspects of living cells, and are thus encoded in every cell's genome. For the creation of artificial cellular systems, genetic information and production of membrane building blocks will need to be coupled in a similar manner. However, natural biochemical reaction networks and membrane building blocks are notoriously difficult to implement in vitro. Here, we utilized amphiphilic elastin-like peptides (ELP) to create self-assembled vesicular st ...[more]

PMID: 30242152

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Project description:Synthetic antibody libraries have proven immensely useful for the de novo isolation of antibodies without the need for animal immunization. Recently, focused libraries designed to recognize particular classes of ligands, such as haptens or proteins, have been employed to facilitate the selection of high-affinity antibodies. Focused libraries are built using V regions encoding combinations of canonical structures that resemble the structural features of antibodies that bind the desired class of ligands and sequence diversity is introduced at residues typically involved in recognition. Here we describe the generation and experimental validation of two different single-chain antibody variable fragment libraries that efficiently generate binders to peptides, a class of molecules that has proven to be a difficult target for antibody generation. First, a human anti-peptide library was constructed by diversifying a scaffold: the human variable heavy chain (V(H)) germ line gene 3-23, which was fused to a variant of the human variable light chain (V(L)) germ line gene A27, in which L1 was modified to encode the canonical structure found in anti-peptide antibodies. The sequence diversity was introduced into 3-23 (V(H)) only, targeting for diversification residues commonly found in contact with protein and peptide antigens. Second, a murine library was generated using the antibody 26-10, which was initially isolated based on its affinity to the hapten digoxin, but also binds peptides and exhibits a canonical structure pattern typical of anti-peptide antibodies. Diversity was introduced in the V(H) only using the profile of amino acids found at positions that frequently contact peptide antigens. Both libraries yielded binders to two model peptides, angiotensin and neuropeptide Y, following screening by solution phage panning. The mouse library yielded antibodies with affinities below 20 nM to both targets, although only the V(H) had been subjected to diversification.

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Towards synthetic cells using peptide-based reaction compartments.

Publications

Towards synthetic cells using peptide-based reaction compartments.

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