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  1. Article: A Cost-Effective

    Schloßhauer, Jeffrey L / Dondapati, Srujan Kumar / Kubick, Stefan / Zemella, Anne

    Bioengineering (Basel, Switzerland)

    2024  Volume 11, Issue 1

    Abstract: Cell-free systems are particularly attractive for screening applications and the production of difficult-to-express proteins. However, the production of cell lysates is difficult to implement on a larger scale due to large time requirements, cultivation ... ...

    Abstract Cell-free systems are particularly attractive for screening applications and the production of difficult-to-express proteins. However, the production of cell lysates is difficult to implement on a larger scale due to large time requirements, cultivation costs, and the supplementation of cell-free reactions with energy regeneration systems. Consequently, the methylotrophic yeast
    Language English
    Publishing date 2024-01-18
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2746191-9
    ISSN 2306-5354
    ISSN 2306-5354
    DOI 10.3390/bioengineering11010092
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  2. Article ; Online: Enriched cell-free and cell-based native membrane derived vesicles (nMV) enabling rapid in-vitro electrophysiological analysis of the voltage-gated sodium channel 1.5.

    Pandey, Yogesh / Dondapati, Srujan Kumar / Kubick, Stefan

    Biochimica et biophysica acta. Biomembranes

    2023  Volume 1865, Issue 5, Page(s) 184144

    Abstract: Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We ... ...

    Abstract Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNa
    MeSH term(s) Cricetinae ; Animals ; Humans ; Cricetulus ; CHO Cells ; Voltage-Gated Sodium Channels ; Membrane Proteins ; Lidocaine
    Chemical Substances Voltage-Gated Sodium Channels ; Membrane Proteins ; Lidocaine (98PI200987)
    Language English
    Publishing date 2023-03-06
    Publishing country Netherlands
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 60-7
    ISSN 1879-2642 ; 1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2618 ; 1879-2650 ; 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
    ISSN (online) 1879-2642 ; 1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2618 ; 1879-2650
    ISSN 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
    DOI 10.1016/j.bbamem.2023.184144
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    In stock of ZB MED Cologne/Königswinter

    Uc I Zs.247: Show issues Location:
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  3. Article: Cell-Free Synthesis and Electrophysiological Analysis of Multipass Voltage-Gated Ion Channels Tethered in Microsomal Membranes.

    Pandey, Yogesh / Dondapati, Srujan Kumar / Wüstenhagen, Doreen / Kubick, Stefan

    Advances in biochemical engineering/biotechnology

    2023  Volume 186, Page(s) 103–120

    Abstract: Cell-free protein synthesis (CFPS) has emerged as a powerful tool for the rapid synthesis and analysis of various structurally and functionally distinct proteins. These include 'difficult-to-express' membrane proteins such as large multipass ion channel ... ...

    Abstract Cell-free protein synthesis (CFPS) has emerged as a powerful tool for the rapid synthesis and analysis of various structurally and functionally distinct proteins. These include 'difficult-to-express' membrane proteins such as large multipass ion channel receptors. Owing to their membrane localization, eukaryotic CFPS supplemented with endoplasmic reticulum (ER)-derived microsomal vesicles has proven to be an efficient system for the synthesis of functional membrane proteins. Here we demonstrate the applicability of the eukaryotic cell-free systems based on lysates from the mammalian Chinese Hamster Ovary (CHO) and insect Spodoptera frugiperda (Sf21) cells. We demonstrate the efficiency of the systems in the de novo cell-free synthesis of the human cardiac ion channels: ether-a-go-go potassium channel (hERG) K
    MeSH term(s) Animals ; Cricetinae ; Humans ; Ether-A-Go-Go Potassium Channels/genetics ; CHO Cells ; Cricetulus ; Heart ; Membrane Proteins
    Chemical Substances Ether-A-Go-Go Potassium Channels ; Membrane Proteins
    Language English
    Publishing date 2023-08-28
    Publishing country Germany
    Document type Journal Article
    ISSN 0724-6145
    ISSN 0724-6145
    DOI 10.1007/10_2023_228
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  4. Article ; Online: Evaluation of the Ion Channel Assembly in a Eukaryotic Cell-Free System Focusing on Two-Pore Domain Potassium Channels K

    Ullrich, Jessica / Ohlhoff, Carsten / Dondapati, Srujan Kumar / Zemella, Anne / Kubick, Stefan

    International journal of molecular sciences

    2023  Volume 24, Issue 7

    Abstract: Oligomeric ion channels are abundant in nature. However, the recombinant expression in cell culture-based systems remains tedious and challenging due to negative side effects, limiting the understanding of their role in health and disease. Accordingly, ... ...

    Abstract Oligomeric ion channels are abundant in nature. However, the recombinant expression in cell culture-based systems remains tedious and challenging due to negative side effects, limiting the understanding of their role in health and disease. Accordingly, in this work, we demonstrate the cell-free synthesis (CFS) as an alternative platform to study the assembly of two-pore domain potassium channels (K
    MeSH term(s) Eukaryota/metabolism ; Potassium Channels, Tandem Pore Domain/genetics ; Potassium Channels, Tandem Pore Domain/metabolism ; Cell-Free System/metabolism ; Dimerization ; Biological Assay
    Chemical Substances Potassium Channels, Tandem Pore Domain
    Language English
    Publishing date 2023-03-27
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2019364-6
    ISSN 1422-0067 ; 1422-0067 ; 1661-6596
    ISSN (online) 1422-0067
    ISSN 1422-0067 ; 1661-6596
    DOI 10.3390/ijms24076299
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  5. Article ; Online: Unraveling the kinetics and pharmacology of human PepT1 using solid supported membrane-based electrophysiology.

    Körner, Alexander / Bazzone, Andre / Wichert, Maximilian / Barthmes, Maria / Dondapati, Srujan Kumar / Fertig, Niels / Kubick, Stefan

    Bioelectrochemistry (Amsterdam, Netherlands)

    2023  Volume 155, Page(s) 108573

    Abstract: The human Peptide Transporter 1 (hPepT1) is known for its broad substrate specificity and its ability to transport (pro-)drugs. Here, we present an in-depth comprehensive study of hPepT1 and its interactions with various substrates via solid supported ... ...

    Abstract The human Peptide Transporter 1 (hPepT1) is known for its broad substrate specificity and its ability to transport (pro-)drugs. Here, we present an in-depth comprehensive study of hPepT1 and its interactions with various substrates via solid supported membrane-based electrophysiology (SSME). Using hPepT1-containing vesicles, we could not identify any peptide induced pre-steady-state currents, indicating that the recorded peak currents reflect steady-state transport. Electrogenic co-transport of H
    MeSH term(s) Humans ; Symporters/metabolism ; Peptide Transporter 1 ; Electrophysiology ; Peptides ; Kinetics
    Chemical Substances Symporters ; Peptide Transporter 1 ; Peptides
    Language English
    Publishing date 2023-09-16
    Publishing country Netherlands
    Document type Journal Article
    ZDB-ID 2010650-6
    ISSN 1878-562X ; 0302-4598 ; 1567-5394
    ISSN (online) 1878-562X
    ISSN 0302-4598 ; 1567-5394
    DOI 10.1016/j.bioelechem.2023.108573
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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1178: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (1.OG)
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  6. Article ; Online: Evaluation of the Ion Channel Assembly in a Eukaryotic Cell-Free System Focusing on Two-Pore Domain Potassium Channels K2P

    Ullrich, Jessica / Ohlhoff, Carsten / Dondapati, Srujan Kumar / Zemella, Anne / Kubick, Stefan

    2023  

    Abstract: Oligomeric ion channels are abundant in nature. However, the recombinant expression in cell culture-based systems remains tedious and challenging due to negative side effects, limiting the understanding of their role in health and disease. Accordingly, ... ...

    Abstract Oligomeric ion channels are abundant in nature. However, the recombinant expression in cell culture-based systems remains tedious and challenging due to negative side effects, limiting the understanding of their role in health and disease. Accordingly, in this work, we demonstrate the cell-free synthesis (CFS) as an alternative platform to study the assembly of two-pore domain potassium channels (K2P) within endogenous endoplasmic reticulum-derived microsomes. Exploiting the open nature of CFS, we investigate the cotranslational translocation of TREK-2 into the microsomes and suggest a cotranslational assembly with typical single-channel behavior in planar lipid-bilayer electrophysiology. The heteromeric assembly of K2P channels is a contentious matter, accordingly we prove the successful assembly of TREK-2 with TWIK-1 using a biomolecular fluorescence complementation assay, Western blot analysis and autoradiography. The results demonstrate that TREK-2 homodimer assembly is the initial step, followed by heterodimer formation with the nascent TWIK-1, providing evidence of the intergroup heterodimerization of TREK-2 and TWIK-1 in eukaryotic CFS. Since K2P channels are involved in various pathophysiological conditions, including pain and nociception, CFS paves the way for in-depth functional studies and related pharmacological interventions. This study highlights the versatility of the eukaryotic CFS platform for investigating ion channel assembly in a native-like environment.

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    Keywords cell-free synthesis (CFS) ; eukaryotic cell-free protein synthesis (CFPS) ; heterodimerization ; ion channel ; K2P ; membrane protein synthesis ; oligomerization ; protein assembly ; TREK-2 ; TWIK-1
    Subject code 572
    Language English
    Publishing country de
    Document type Article ; Online
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  7. Article ; Online: Cell-Free Protein Synthesis: A Promising Option for Future Drug Development.

    Dondapati, Srujan Kumar / Stech, Marlitt / Zemella, Anne / Kubick, Stefan

    BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy

    2020  Volume 34, Issue 3, Page(s) 327–348

    Abstract: Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing ... ...

    Abstract Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development.
    MeSH term(s) Animals ; CHO Cells ; Cell-Free System ; Cricetulus ; Drug Development/methods ; Membrane Proteins/biosynthesis ; Microsomes ; Protein Biosynthesis ; Protein Folding ; Receptors, G-Protein-Coupled/biosynthesis
    Chemical Substances Membrane Proteins ; Receptors, G-Protein-Coupled
    Keywords covid19
    Language English
    Publishing date 2020-03-20
    Publishing country New Zealand
    Document type Journal Article ; Review
    ZDB-ID 1364202-9
    ISSN 1179-190X ; 1173-8804
    ISSN (online) 1179-190X
    ISSN 1173-8804
    DOI 10.1007/s40259-020-00417-y
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    In stock of ZB MED Cologne/Königswinter

    Zs.A 4112: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (2.OG)
    ab Jg. 2022: Lesesaal (EG)

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  8. Article ; Online: Functional Analysis of Membrane Proteins Produced by Cell-Free Translation.

    Dondapati, Srujan Kumar / Wüstenhagen, Doreen A / Kubick, Stefan

    Methods in molecular biology (Clifton, N.J.)

    2017  Volume 1685, Page(s) 171–186

    Abstract: Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are ... ...

    Abstract Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are scalable from 20 μL up to several mL, faster and suitable for the high-throughput protein production. Here, we present two cell-free systems derived from Escherichia coli (E. coli) and Spodoptera frugiperda (Sf21) lysates. In the case of the E. coli cell-free system, nanodiscs are used for the solubilization and purification of membrane proteins. In the case of the Sf21 system, endogenous microsomes with an active translocon complex are present within the lysates which facilitate the incorporation of the bacterial potassium channel KcsA within the microsomal membranes. Following cell-free synthesis, these microsomes are directly used for the functional analysis of membrane proteins.
    MeSH term(s) Animals ; Cell-Free System ; Escherichia coli/cytology ; Escherichia coli/metabolism ; Membrane Proteins/biosynthesis ; Microsomes/metabolism ; Potassium Channels/biosynthesis ; Sf9 Cells
    Chemical Substances Membrane Proteins ; Potassium Channels
    Language English
    Publishing date 2017-10-30
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-4939-7366-8_10
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  9. Article ; Online: Erratum to: Functional Analysis of Membrane Proteins Produced by Cell-Free Translation.

    Dondapati, Srujan Kumar / Wüstenhagen, Doreen A / Kubick, Stefan

    Methods in molecular biology (Clifton, N.J.)

    2017  Volume 1685, Page(s) E1

    Language English
    Publishing date 2017-11-16
    Publishing country United States
    Document type Journal Article ; Published Erratum
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-4939-7366-8_21
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  10. Article ; Online: Targeted esterase-induced dye (TED) loading supports direct calcium imaging in eukaryotic cell-free systems.

    Dhandapani, Priyavathi / Dondapati, Srujan Kumar / Zemella, Anne / Bräuer, Dennis / Wüstenhagen, Doreen Anja / Mergler, Stefan / Kubick, Stefan

    RSC advances

    2021  Volume 11, Issue 27, Page(s) 16285–16296

    Abstract: Calcium imaging is an important functional tool for analysing ion channels, transporters and pumps for drug screening in living cells. Depicted eukaryotic cell-free systems utilize microsomes, derived from the endoplasmic reticulum to incorporate the ... ...

    Abstract Calcium imaging is an important functional tool for analysing ion channels, transporters and pumps for drug screening in living cells. Depicted eukaryotic cell-free systems utilize microsomes, derived from the endoplasmic reticulum to incorporate the synthesized membrane proteins-like ion channels. Carboxylesterase is required to cleave the acetoxymethyl ester moiety of the chemical calcium indicators in order to ensure its immobility across the endoplasmic reticulum membrane. Absence or an inadequate amount of carboxylesterase in the endoplasmic reticulum of different eukaryotic cells poses a hindrance to perform calcium imaging in microsomes. In this work, we try to overcome this drawback and adapt the cell-based calcium imaging principle to a cell-free protein synthesis platform. Carboxylesterase synthesized in a
    Language English
    Publishing date 2021-05-04
    Publishing country England
    Document type Journal Article
    ISSN 2046-2069
    ISSN (online) 2046-2069
    DOI 10.1039/d0ra08397f
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