Top PDF A User’s Guide to Cell-Free Protein Synthesis

A User’s Guide to Cell-Free Protein Synthesis

A User’s Guide to Cell-Free Protein Synthesis

Within the last year, the Jewett, Church, and Siemann-Herzberg laboratories have each separately developed a CFPS platform based upon Vibrio natriegens [10,170,171]. With its doubling time being the shortest of all known organisms, its high rate of protein synthesis, and high metabolic efficiency, this platform has potential to be an ideal candidate for CFPS [170]. In addition to its unique doubling time, Vibrio natriegens extract preparation requires a stationary phase harvest for the highest translational efficiency in a CFPS platform. Typically, CFPS extracts are harvested in a tight window during the mid-exponential phase to maximize translational efficiency. However, the Vibrio natriegens extract allows a great amount of flexibility for the user to “set and forget” the culture for a stationary phase harvest where ribosome production is thought to be lowest among other microorganisms [10]. Another advantage to extract preparation for this platform is its high resistance to damage via over-lysis. Additionally, it is relatively agnostic to lysis buffer resuspension volume. Together, these allow for inexperienced CFPS users to easily generate robust extract [10]. In addition, the V. natriegens platform generates a very high volume of extract compared to the standard E. coli platform, allowing for 8–12 mL of active lysate per L of culture compared to just 1–3 mL/L for E. coli when grown in shake flasks and lysed by sonication [10]. V. natriegens extract has even been shown to maintain 100% of activity after one week of storage at room temperature post-lyophilization in the presence of trehalose [10]. Although this platform appears to be promising in terms of flexibility and scale of extract preparation, very few applications have been proposed. Aside from reporter proteins being expressed, the Jewett laboratory has demonstrated the successful synthesis of a series of antimicrobial peptides using this platform [10].
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Synthetic Biology Goes Cell-Free

Synthetic Biology Goes Cell-Free

Molecular research has highlighted the importance of proteinprotein interactions and the resulting complexes that these interactions can generate. Whether it is for the biophysical study of these complexes or as vehicles for new therapeutic delivery (e.g., virus-like scaffolds for vaccines), there is a growing need for developing robust tools aimed at synthesis of such complexes. As in the case of membrane proteins, CFS have also demonstrated higher yields, compared to in vivo strategies, in the pro- duction of macromolecular assemblies such as virus-like particles (VLPs) [109]. Groundbreaking work by the Swartz group, demonstrating the cell-free expression of hepatitis B core antigen VLP (2 subunits) [91] in an E. coli-based cell-free system, opened the door to other re- searchers expressing a variety of macromolecular assem- blies including the E. coli RNA polymerase (5 subunits) [118] and an ATP synthase (25 subunits) [113]. Earlier work with reticulocyte lysate had also demonstrated cell- free expression of the human T-cell receptor (7 sub- units) [119]. Remarkably, a number of bacteriophages have now also been successfully expressed in CFS, in- cluding the T4 phage, which structurally contains 1500 proteins from 50 genes [56, 102–104] (Fig. 3).
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Cell-Free Synthesis of Encephalomyocarditis Virus

Cell-Free Synthesis of Encephalomyocarditis Virus

tions (Fig. 2C), in sharp contrast to the broad range observed for translation. Lowering the magnesium salt concentration to 2 mM resulted in almost no RNA synthesis (Fig. 2C) while causing only marginal inhibition of translation (data not shown). An EMCV RNA dose-response experiment showed that saturation of RNA synthesis is reached at a surprisingly low level of input RNA (5 ␮g/ml; Fig. 2D). Higher RNA concentrations (10 to 30 ␮g/ml) markedly inhibited RNA syn- thesis. Also surprising was that this inhibition occurred at RNA concentrations which stimulated rather than inhibited viral translation (Fig. 1B). A plausible explanation is that a viral protein(s), when accumulated to a certain level, can inhibit RNA replication. For example, RNA interaction with the structural proteins would engage it in a packaging complex and would preclude replication. Alternatively, EMCV RNA excess may sequester a host factor(s), which would facilitate RNA replication (3, 9). It would be interesting to determine whether the negative-feedback mechanism for regulation of RNA rep- lication by virus-specific products governs the switch from rep- lication to encapsidation of the viral RNA in vivo.
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Tryptic peptide analyses of polypeptides generated by premature termination of cell-free protein synthesis allow a determination of the Rauscher leukemia virus gag gene order.

Tryptic peptide analyses of polypeptides generated by premature termination of cell-free protein synthesis allow a determination of the Rauscher leukemia virus gag gene order.

Tryptic Peptide Analyses of Polypeptides Generated by Premature Termination of Cell-Free Protein Synthesis Allow a Determination of the Rauscher Leukemia Virus gag Gene Order Received fo[r]

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The influence of the penetrating cryoprotectors on protein synthesis in the cell-free system of the rat liver

The influence of the penetrating cryoprotectors on protein synthesis in the cell-free system of the rat liver

Гель-фильтрация постмитохондриального супернатанта через колонку с сефадексом G-25. Для гель­фильтрации фракции S 15 готовили колонку из сефадекса G­25 2,5х20 см. Колонка заполнялась и хранилась в бидистиллирован­ ной воде в течение 6 часов для полного на­ бухания зерен сефадекса. Перед нанесением препарата колонку уравновешивали колоноч­ ным буфером в мМ: 85 – KCl, 3,5 – MgCl 2 , 30 – трис­HCl (рН 7,5). Фракцию S 15 собира­ ли в отдельную емкость сразу же после выхода расчетного колоночного объема буфера. Пос­ ле прохождения через колонку с сефадексом, фракция S 15 содержала от 13 до 20 мг/мл про­ теина.
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Protein synthesis in a cell free human reticulocyte system: ribosome function in thalassemia

Protein synthesis in a cell free human reticulocyte system: ribosome function in thalassemia

In the present investigation, a cell free system for protein synthesis has been developed from human reticulocytes and this latter hypothesis tested further by comparing the capacity for[r]

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Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

The virus was propagated twice in suckling mouse brain, isolated, plaque purified four times on L929 cells, and then grown in Sac- cells at low multiplic10 Downloaded from http://jvi.asm[r]

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Protein Synthesis in Cell-Free Systems: an Effect of Interferon

Protein Synthesis in Cell-Free Systems: an Effect of Interferon

Stimulation of amino acid incorporation by EMC RNA with untreated "74S" ribosomes from control chick embryo fibroblasts CEF, U and CEF which had been exposed to 35 0 and 140 A units of i[r]

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Stögbauer, Tobias Roland
  

(2012):


	Experiment and quantitative modeling of cell-free gene expression dynamics.


Dissertation, LMU München: Fakultät für Physik

Stögbauer, Tobias Roland (2012): Experiment and quantitative modeling of cell-free gene expression dynamics. Dissertation, LMU München: Fakultät für Physik

Cell-free systems were originally developed as a convenient means to synthesize proteins that are hard to produce in cells like toxic proteins. Nevertheless due to their comparative simplicity and ease of modification they also exhibit great potential as in vitro model plat- forms for transcription/translation studies. This however necessitates knowledge of kinetic parameters, expected protein yield, necessary template DNA concentration and reaction speed of the particular cell-free system which is generally not provided. Here a procedure of how to specify gene expression dynamics of a cell-free system through a combination of a set of calibration experiments and a mathematical model is established. The presented model concurrently fits the entirety of the data with one set of eight parameters. This was achieved by expanding the classical rate equation scheme as it is known from work in vivo (chapter 5) in two key points: first, a Michaelis-Menten like ansatz to account for saturation at high DNA concentrations and second, finite resource pools for transcription and translation. In this manner the model presented here adequately captures both the early rising phase and the late plateau phase of cell-free protein synthesis. It does so con- sistently over five orders of magnitude of DNA concentration with one set of fit parameters. After calibrating the model to the respective cell-free system used in an experiment the model predictively describes time courses of mRNA and protein synthesis as a function of template concentration and experimental timing. The fact that here a more general model (Hill formalism for protein synthesis and two different decay processes for transcription and translation resources) was reduced to a simpler form solely through parameter optimization of simulated values to experimental data is quite remarkable. In principle a more general formalism should result in a better fit because of the trade-off between model simplicity and fit quality. Here the experimental data seem to be well suited for discriminating the models and support the simplification. Note however that in the case of significant RNAse or protease activity in a particular cell-free system the more general expressions (4.6) and (4.7) for T sR and T lR time courses have to be tested.
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Cell free expression and in meso crystallisation of an integral membrane kinase for structure determination

Cell free expression and in meso crystallisation of an integral membrane kinase for structure determination

Cell-free expression is extremely efficient in that milligrams of protein can be produced overnight in 1 mL of reaction mix. Further, because it is an open system the protein can be harvested directly without the need for all that goes with culturing live cells and isolating membranes or inclusion bodies from harvested biomass. The cell-free synthesised protein is relatively pure to begin with requiring, in the case of DgkA and EmrE [32], little more than sequential nickel affinity and size-exclusion chromatographic steps to generate protein of a quality suitable for crystal structure work. Indeed, with a different DgkA construct, Δ4 DgkA [33], diffraction quality crystals were obtained using protein that had only been purified by nickel affinity chromatography (unpublished observations). It is, of course, necessary in the cell-free system of the precipitate type to solubilise the precipitated protein and to refold it as a prelude to purification and crystallisation. The option is always there however, to perform expression in the presence of detergent micelles [34], liposomes [35] or nanodiscs [36], which act as membrane mimetic receptacles for the nascent protein, with a view to diverting it, partly at least, away from aggregation. These alternative approaches however, usually require additional steps where the ‘reconstituted’ protein is isolated from aggregated material and the protein is removed or exchanged from its membrane mimetic and reformulated in preparation for crystallisation screening.
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USER GUIDE

USER GUIDE

• Start the Visual Voice Mail application (see “To start the Visual Voice Mail application” on page 36) while docked in 5230 IP Appliance.... 38 Revision A 5230 IP Appliance User Guide.[r]

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User Guide

User Guide

In the left ear wearing style, with the corded side of the receiver facing you, hold the headset in your left hand between your thumb and index finger with the boom pointing to the left.[r]

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User Guide and Administrator Guide

User Guide and Administrator Guide

Press the Menu button, choose Admin Setup and press the Select button, enter passcode (if required) and press the ENTER soft key.. Choose Diagnostics using the Scrolling buttons, [r]

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USER GUIDE

USER GUIDE

Most services have customizable options to allow you to personalize your phone to best meet your needs. The 4 key provides access to all customizable options. You can choose to protect your options with a password, if desired. Use the display screen to view and access the options. See the specific procedures in this guide for detailed information about how to customize each option.

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User Guide

User Guide

Call forwarding, call waiting, and send own number are network services available through your service provider. These features do not appear in your phone’s Network services menu until you save the related feature code(s) given to you by your service provider. After you save the feature activation or cancellation code, the feature appears in the phone’s menu and you can use the menu to turn the feature on or off. Note: Feature codes can be saved only when your primary phone number (NAM 1) is selected. See page 33.

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User Guide

User Guide

The term wireless phone refers here to hand-held wireless phones with built-in antennas, often called cell mobile or PCS phones. These types of wireless phones can expose the user to measurable radiofrequency energy (RF) because of the short distance between the phone and the user’s head. These RF exposures are limited by Federal Communications Commission safety guidelines that were developed with the advice of FDA and other federal health and safety agencies. When the phone is located at greater distances from the user, the exposure to RF is drastically lower because a person's RF exposure decreases rapidly with increasing distance from the source. The so-called cordless phones; which have a base unit connected to the telephone wiring in a house, typically operate at far lower power levels, and thus produce RF exposures far below the FCC safety limits.
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User Guide

User Guide

Congratulations on your purchase of the GN Netcom Contour LX-G headset. This guide contains instructions for the Contour LX-G headset. To derive maximum benefit from this product, please take a few minutes to review this user’s guide. If after reviewing this guide you have any questions concerning the Contour LX-G headset, please call your distributor or contact GN Netcom, Inc. Customer Service at 800-826-4656.

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USER GUIDE

USER GUIDE

626 This feature enables an extension user to "pull" calls from their usual extension to the extension that they are currently working at. This feature ensures that calls continue to follow an extension user when working at various desks within the office.

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User Guide

User Guide

For these applications, the headset is equipped with a Quick Disconnect plug which mates to a Quick Disconnect jack on the amplifier.. Follow the instructions supplied with the amplifier[r]

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USER GUIDE

USER GUIDE

Feature Keys 7RGLVSOD\LQIRUPDWLRQDERXWDNH\ 3UHVV 683(5.(< 3UHVVDSHUVRQDONH\ 3UHVV 683(5.(< 7RSURJUDPDSHUVRQDONH\ 3UHVV 6XSHU.H\. 3UHVV UHSHDWHGO\XQWLO3(5621$/.(<6"LV[r]

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