Protein Incorporation in Giant Lipid Vesicles under Physiological Conditions

Shaklee, Paige M.; Semrau, Stefan; Malkus, Maurits; Kubick, Stefan; Dogterom, Marileen; Schmidt, Thomas

doi:10.1002/cbic.200900669

Cited by 44 publications

(48 citation statements)

References 33 publications

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“…According to the characteristics of the protein of interest, these so-called microsomes can then be used as microcontainers enabling the enrichment of the translocated target protein. They also serve as the prerequisite for PTMs such as glycosylation, lipidation, and signal peptide cleavage [11,12,26]. Furthermore, insect vesicles may have a positive effect on the formation of disulfide bonds in proteins inasmuch as they mimic the appropriate environment present in living cells.…”

Section: Using Microsomal Vesicles As Protein Microcontainersmentioning

confidence: 99%

“…This strategy has been successfully performed by using a homogeneous cell-free translation system based on insect cell lysates that contain microsomal vesicles derived from the ER [11]. As reported in the literature, these membrane vesicles can be manipulated regarding their size [26] and physicochemical properties. Such a manipulation allows the implementation of membrane vesicles in technical systems such as biochips or microarrays, opening up new strategies to screen for pharmacologically relevant target proteins.…”

Section: Introductionmentioning

confidence: 99%

“…The enzymatic activity of the biological membranes survives this process as protein translocation and membrane protein insertion have been reported. Using this type of lysate PTMs such as glycosylation, lipidation, phosphorylation, signal peptide cleavage, and disulfide bond formation can be performed on de novo synthesized proteins [11,12,[25][26][27][28].…”

Section: Introductionmentioning

confidence: 99%

“…To date, the expression of many functional and structural divergent membrane proteins in the insect cell-free system has been reported [11,12,26]. As examples, we present a repertory of integral membrane proteins ranging from type I membrane proteins (Hb-EGF and EGFR) across a six-transmembrane ion-channel (Aqp1) to a seven-transmembrane G-protein coupled receptor (ETB; Table 1).…”

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confidence: 99%

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Cell-Free Systems: Functional Modules for Synthetic and Chemical Biology

Stech¹,

Brödel²,

Quast³

et al. 2013

Fundamentals and Application of New Bioproduction Systems

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: The main goal of cell-free protein synthesis is to produce correctly folded and functional proteins in reasonable amounts for further downstream applications. Especially for eukaryotic proteins, functionality is often directly linked to the presence of posttranslational modifications. Thus, it is of highest interest to develop novel cell-free expression systems that enable the synthesis of posttranslationally modified proteins. Here we present recent advances for the synthesis of glycoproteins, proteins containing disulfide bridges, membrane proteins, and fluorescently labeled proteins. The basis for the expression of these difficult-to-express target proteins is a translationally active cell extract which can be prepared from eukaryotic cell lines such as Spodoptera frugiperda 21 (Sf21) and Chinese hamster ovary (CHO) cells. Due to a very mild lysate preparation procedure, microsomal vesicles derived from the endoplasmic reticulum (ER) can be maintained in the eukaryotic lysate. These vesicles are translocationally active and serve as functional modules facilitating protein translocation and enrichment as well as posttranslational modification of de novo synthesized proteins. In particular, for the synthesis of membrane proteins microsomal vesicles are the essential prerequisite for the insertion of the desired protein into a biologically active membrane scaffold providing a natural environment. We anticipate that the use of such translationally active eukaryotic cell lysates containing translocationally active vesicles may solve a large number of problems still persistent when expressing eukaryotic proteins in vitro.

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Section: Using Microsomal Vesicles As Protein Microcontainersmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Cell-Free Systems: Functional Modules for Synthetic and Chemical Biology

Stech¹,

Brödel²,

Quast³

et al. 2013

Fundamentals and Application of New Bioproduction Systems

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“…Zusätzlich können GPCR-enthaltende Mikrosomen zu großen Vesikeln (giant unilamellar vesicles, GUV) umgebaut werden und bilden damit ein System für mikroskopische Einzelmolekülanalysen zur Charakterisierung der Zielproteine [4,6].…”

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Membranproteinsynthese: Zellfrei geht’s schneller!

et al. 2014

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Difficult to express membrane proteins represent an increasing amount of therapeutic molecules. Considerable optimization is often required for downstream applications such as assay development and functional characterization. Cell-free systems emerged as powerful tools for the synthesis of structurally and functionally divergent membrane proteins. Vesicle-based eukaryotic cell-free systems enable co-translational protein translocation and posttranslational modifications. Hence, these systems provide a multitude of options for membrane protein studies.

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Giant Vesicles: Preparations and Applications

et al. 2010

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There is considerable interest in preparing cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biological cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chemical compounds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible preparation of giant vesicles are available. The key concepts of currently known methods for preparing giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.

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Protein Incorporation in Giant Lipid Vesicles under Physiological Conditions

Cited by 44 publications

References 33 publications

Cell-Free Systems: Functional Modules for Synthetic and Chemical Biology

Cell-Free Systems: Functional Modules for Synthetic and Chemical Biology

Membranproteinsynthese: Zellfrei geht’s schneller!

Giant Vesicles: Preparations and Applications

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