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      Cell-free Production : System Development 

      Cell-Free Synthesis and Electrophysiological Analysis of Multipass Voltage-Gated Ion Channels Tethered in Microsomal Membranes

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          There is no author summary for this book yet. Authors can add summaries to their books on ScienceOpen to make them more accessible to a non-specialist audience.

          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 V11.1 and the voltage-gated sodium channel hNa V1.5.

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          Calcium and Excitation-Contraction Coupling in the Heart

          David A. Eisner, Jessica L Caldwell, Kornel Kistamas … (2017)
          Cardiac contractility is regulated by changes in intracellular Ca concentration ([Ca2+]i). Normal function requires that [Ca2+]i be sufficiently high in systole and low in diastole. Much of the Ca needed for contraction comes from the sarcoplasmic reticulum and is released by the process of calcium-induced calcium release. The factors that regulate and fine-tune the initiation and termination of release are reviewed. The precise control of intracellular Ca cycling depends on the relationships between the various channels and pumps that are involved. We consider 2 aspects: (1) structural coupling: the transporters are organized within the dyad, linking the transverse tubule and sarcoplasmic reticulum and ensuring close proximity of Ca entry to sites of release. (2) Functional coupling: where the fluxes across all membranes must be balanced such that, in the steady state, Ca influx equals Ca efflux on every beat. The remainder of the review considers specific aspects of Ca signaling, including the role of Ca buffers, mitochondria, Ca leak, and regulation of diastolic [Ca2+]i.
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            Cell-free protein synthesis: applications come of age.

            Erik D Carlson, Rui Gan, C Eric Hodgman … (2012)
            Cell-free protein synthesis has emerged as a powerful technology platform to help satisfy the growing demand for simple and efficient protein production. While used for decades as a foundational research tool for understanding transcription and translation, recent advances have made possible cost-effective microscale to manufacturing scale synthesis of complex proteins. Protein yields exceed grams protein produced per liter reaction volume, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and reaction scale has reached the 100-liter milestone. These advances have inspired new applications in the synthesis of protein libraries for functional genomics and structural biology, the production of personalized medicines, and the expression of virus-like particles, among others. In the coming years, cell-free protein synthesis promises new industrial processes where short protein production timelines are crucial as well as innovative approaches to a wide range of applications. Copyright © 2011 Elsevier Inc. All rights reserved.
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              Mapping the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin

              Markus Sällman Almén, Karl Nordström, Robert Fredriksson … (2009)
              Background Membrane proteins form key nodes in mediating the cell's interaction with the surroundings, which is one of the main reasons why the majority of drug targets are membrane proteins. Results Here we mined the human proteome and identified the membrane proteome subset using three prediction tools for alpha-helices: Phobius, TMHMM, and SOSUI. This dataset was reduced to a non-redundant set by aligning it to the human genome and then clustered with our own interactive implementation of the ISODATA algorithm. The genes were classified and each protein group was manually curated, virtually evaluating each sequence of the clusters, applying systematic comparisons with a range of databases and other resources. We identified 6,718 human membrane proteins and classified the majority of them into 234 families of which 151 belong to the three major functional groups: receptors (63 groups, 1,352 members), transporters (89 groups, 817 members) or enzymes (7 groups, 533 members). Also, 74 miscellaneous groups with 697 members were determined. Interestingly, we find that 41% of the membrane proteins are singlets with no apparent affiliation or identity to any human protein family. Our results identify major differences between the human membrane proteome and the ones in unicellular organisms and we also show a strong bias towards certain membrane topologies for different functional classes: 77% of all transporters have more than six helices while 60% of proteins with an enzymatic function and 88% receptors, that are not GPCRs, have only one single membrane spanning α-helix. Further, we have identified and characterized new gene families and novel members of existing families. Conclusion Here we present the most detailed roadmap of gene numbers and families to our knowledge, which is an important step towards an overall classification of the entire human proteome. We estimate that 27% of the total human proteome are alpha-helical transmembrane proteins and provide an extended classification together with in-depth investigations of the membrane proteome's functional, structural, and evolutionary features.
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                Author and book information

                Book Chapter
                Publication date (Print): 2023
                Publication date (Online): August 29 2023
                Pages: 103-120
                DOI: 10.1007/10_2023_228
                SO-VID: 2ef3dc83-237f-4d0c-bf85-d95a57e77b9b
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