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Download The Pharmacology Of Functional, Biochemical, And Recombinant Receptor Systems 2000
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Details Collect From N Order a copy Copyright or permission restrictions may apply. We will contact you if necessary. To learn more about Copies Direct watch this short online video. To further substantiate this, we treated cells with FTI, a selective farnesyl-transferase inhibitor Appels et al. A Kinetochore localization of unmodified recombinant Spindly depends on cellular farnesylation.
Panels show representative images of cells transduced with unfarnesylated mCherry-Spindly in nocodazole-treated HeLa cells. Mutation of Cys to alanine prevents farnesylation upon EP and reduces KT levels of the delivered protein. Controls cells were electroporated with mCherry. Insets represent magnifications of the indicated kinetochores. C In vitro farnesylation of mCherry-Spindly before EP bypasses the need of cellular farnesylation to achieve kinetochore localization.
Although we do not provide formal proof, the persistent treatment with FT inhibitors in this experiment suggests that Spindly F may be the only residual pervasively farnesylated protein in the target cells.
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Together, these findings substantiated the notion that pre-farnesylation of recombinant Spindly bypassed the need for endogenous farnesyl-transferase activity for proper kinetochore localization in HeLa cells. The modification of biological macromolecules through the introduction of new functionalities has progressed at a tremendous pace in recent years, enriching the palette of tools for biological investigation with a wide spectrum of functionalities. For instance, genetic code expansion, an approach that allows to introduce chemical functionalities into proteins, ranging from synthetic, photostable fluorescent dyes with high quantum yields to reactive groups e.
With rapid growth of chemical and synthetic biology, the demand for robust methods to deliver modified synthetic or semi-synthetic macromolecules into cells is growing Lienert et al. While electroporation has been long recognized for its potential as a delivery approach, questions have been raised regarding the degree of its invasiveness and damage inferred on cellular structures, most notably membranes Torchilin, Here, we have assessed the suitability of batch EP for delivering recombinant proteins into living mammalian cells for various applications in cell biology.
In comparison to previous studies that focused on cytosolic proteins devoid of specific localization, our focus was on kinetochores, which are small, discrete subcellular structures. This was crucial, because it enabled a detailed assessment of the effective degree of functional complementation of the electroporated samples. Our results identify EP as a rapid, efficient, and semi-quantitative technique that enables the delivery into various cultured mammalian cell lines of proteins of variable mass and hydrodynamic radius, including the highly elongated NDC80C.
Importantly, we provide clear evidence that the delivered proteins remain largely structurally and functionally intact after delivery, as witnessed by their correct kinetochore localization and, where applicable, ability to complement siRNA-based depletion, dominant negative effects, and immunoprecipitation with endogenous interacting partners. Thus, in addition to its ease of application, EP is very attractive because it allows delivery in sufficiently large cohorts of cells, and is therefore compatible with cell biochemistry, as clearly shown here.
The Pharmacology of Functional, Biochemical, and Recombinant Receptor Systems - Semantic Scholar
We envision EP to be generally compatible with many macromolecular interaction approaches, from mass spectrometry to immunoprecipitation. If combined with suitable chemical modifications of the probes, for instance by introduction of acutely activatable crosslinking groups, EP may become a method of choice for the identification of elusive binding partners for instance, low affinity substrates of enzymes with high spatial and temporal resolution.
Here, we have demonstrated how the introduction of a farnesyl chain in vitro rescued the kinetochore localization of Spindly in cells treated with a farnesyl transferase inhibitor. In the future, this approach may be extended to other lipid modifications, and may allow monitoring the behavior of only one or a few modified proteins at the time. Another obvious advantage of in vitro manipulations of macromolecules is that it allows precision labeling with small fluorescent dyes, potentially bypassing limits associated with tagging target proteins with bulky genetically encoded fluorescent proteins.
In summary, we envision that EP has the potential to become a method of choice for delivery of synthetic or semi-synthetic proteins into the cellular environment. Between 2 and 3 million cells per electroporation were used in this study. Volumes and protein-buffer ratios may be adjusted according to the purpose of the experiment and depending on protein solubility. Following one further wash in PBS and centrifugation, the cell pellet was re-suspended in complete imaging medium without antibiotics and transferred to a cell-imaging plate Ibidi.
Cells were returned to the incubator and allowed to recover for a minimum of 4 hr. After recovery, cells were either analyzed by fluorescence microscopy imaging or processed to generate protein extracts to be used for either immunoprecipitation analysis or western blotting. EP with the Amaxa Nucleofecto I system was performed according to the reported protocol Theillet et al.
EP conditions described above gave satisfactory results for all the cell types and proteins tested. Only a small fraction of the tested recombinant proteins proved unsuitable for EP delivery.
This was due to formation of major intracellular aggregates or because the protein samples failed to localize correctly within the cell. Cell-specific and protein-specific optimization of such parameter should be always carried out in order to achieve the best trade-off between cell viability and efficient protein delivery. The following are some of the important factors to consider during optimization of protein delivery: 1 In our work, we invariably used protein samples of great purity and homogeneity high monodispersity and solubility.
Although we did not systematically analyze the relationship between protein homogeneity and efficiency of EP, we suspect that protein sample quality is an important factor. All recombinant proteins used in this study were of human origin.
Table of contents
Construct for insect cell expression exploited the MultiBac baculovirus expression system Bieniossek et al. Baculovirus was amplified through three rounds of amplification and used to infect Tnao38 cells. Cells infected with virus were cultured for 72 hr before harvesting. Elution of bound protein was achieved by a linear gradient 25— mM NaCl in 25 column volumes. CFP-Mis12C was purified by a three-step protocol, as described previously for the non-fluorescent version Petrovic et al.
Soluble lysates were passed over a 5 ml Ni-NTA column and, after washing with 20 column volumes buffer A, the proteins were eluted by adding mM imidazole to buffer A. Cell lines were not further authenticated.
Cells used in this study are regularly checked for mycoplasma contamination and test negative. Unless differently indicated, the microtubule-depolimerising drug nocodazole was used at 3. To generate mitotic populations for immunoprecipitation experiments after EP, cells were treated with nocodazole for 16 hr.
The following antibodies were used for the western blot analysis in this study: anti-Bub1 rabbit polyclonal; Abcam; , anti-Hec1 human Ndc80; mouse clone 9G3. Fluorescence intensity from protein extracts derived from a known number of electroporated cells were measured and plotted against a calibration curve generated with defined concentrations of recombinant mCherry.
Bar graphs show average intracellular concentrations and SD for two independent experiments in which every concentration was analysed in duplicate. For viability assays, the percentage of viable cells was measured by Trypan Blue staining followed by automatic counting of viable cells using the Countess automated cell counter Thermo Fisher. Each sample was counted twice and values showed in figures represents the average of these counts.
Secondary antibodies were HRP-conjugated anti-mouse or anti-rabbit Sigma, , dilutions. A correction factor of 0.
For this study, the following microscopes were used. Images were acquired as Z sections using Slidebook Software six from Intelligent Imaging Innovations and converted into maximal-intensity-projection TIFF files for illustrative purposes. Images were acquired as Z sections using the softWoRx software from Deltavision and converted into maximal-intensity-projection TIFF files for illustrative purposes. The microscope was also equipped with an Eppendorf Transjector and an Eppendorf Micromanipulator DNA was stained with 0. Quantification of kinetochore signals was performed on unmodified bit z-series images using Imaris nine software Bitplane.
Measurements were plotted with GraphPad Prism 6. After electroporation cells were allowed to recover overnight and then treated with nocodazole for imaging on the Mariana system. Following EP, cells recovered overnight before being processed for either western blotting analysis or imaged lived on the Deltavision system. Mitotic duration, with or without nM nocodazole, was assessed by DNA morphology.