Reconstitution of Drosophila and human chromatins by wheat germ cell-free co-expression system

Abstract Background Elaboration of the epigenetic regulation of chromatin is a long-standing aim in molecular and cellular biology. Hence, there is a great demand for the development of in vitro methods to reconstitute chromatin that can be used directly for biochemical assays. The widely used wheat germ cell-free protein expression method provides broad applications to investigate the function and structure of eukaryotic proteins. Such advantages, including high translation efficiency, flexibility, and possible automatization, are beneficial for achieving native-like chromatin substrates for in vitro studies. Results We describe a novel, single-step in vitro chromatin assembly method by using the wheat germ cell-free protein synthesis. We demonstrated that both Drosophila and human chromatins can be reconstituted in the course of the in vitro translation of core histones by the addition of chromatin assembly factors, circular plasmid, and topoisomerase I in an ATP-dependent manner. Drosophila chromatin assembly was performed in 4 h at 26 °C, in the presence of premixed mRNAs encoding the core histones, dAcf1/dISWI chromatin remodeling complex, and nucleosome assembly protein, dNAP1. Similarly, the human chromatin was assembled by co-expressing the human core histones with Drosophila chromatin remodeling factor, dISWI, and chromatin chaperone, dNLP, for 6 h at 26 °C. The presence of reconstituted chromatin was monitored by DNA supercoiling assay, also the regular spacing of nucleosomes was assessed by Micrococcal nuclease assay. Furthermore, Drosophila linker histone H1-containing chromatin was reconstituted, affirming that the in vitro assembled chromatin is suitable for downstream applications. Conclusions The method described in this study allows the assembly of Drosophila and human chromatins, possibly in native-like form, by using a wheat germ cell-free protein expression. Although both chromatins were reconstituted successfully, there were unexpected differences with respect to the required ratio of histone-coding mRNAs and the reaction time. Overall, our new in vitro chromatin reconstitution method will aid to characterize the unrevealed structure, function, and regulation of chromatin dynamics.

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A novel family of expression vectors with multiple affinity tags for wheat germ cell-free protein expression

BMC Biotechnology ◽

10.1186/s12896-020-00610-5 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 1

Author(s):

Szilvia Krisztina Nagy ◽

Brigitta Margit Kállai ◽

Judit András ◽

Tamás Mészáros

Keyword(s):

Protein Expression ◽

Germ Cell ◽

Cleavage Site ◽

Wheat Germ ◽

In Vitro Translation ◽

Affinity Tags ◽

Free Protein ◽

Target Proteins ◽

Interaction Studies

Abstract Background Cell-free protein expression has become a widely used alternative of in vivo, cell-based systems in functional and structural studies of proteins. The wheat germ-based method outstands from the commercially available eukaryotic in vitro translation systems by its flexibility, high translation efficiency and success rate of properly folded eukaryotic protein synthesis. The original T7 promoter containing pEU3-NII vector was improved previously by addition of a ligation-independent cloning site, His6- and GST-tags, and a TEV protease cleavage site to facilitate the creation of recombinant plasmids, permit affinity purification, and enable production of purified, tag-free target proteins, respectively. Results Here, we describe a further development of pEU3-NII vector by inserting the rare-cutting, NotI restriction enzyme cleavage site to simplify vector linearization step prior to in vitro transcription. Additionally, His12, FLAG, and Halo affinity tag coding vectors have been created to increase detection sensitivity, specificity of interaction studies, and provide covalently linkable ligands for pull-down assays, respectively. Finally, the presented GST-His6, and GST-biotin double-tagging vectors could broaden the range of possibilities of protein-protein interaction studies. Conclusions The new generation of pEU3-NII vector family allows a more rapid production of translationally active mRNA and wheat germ cell-free expression of target proteins with a wide variety of affinity tags thus enables designing flexible and diverse experimental arrangement for in vitro studies of proteins.

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Dual Roles of p300 in Chromatin Assembly and Transcriptional Activation in Cooperation with Nucleosome Assembly Protein 1 In Vitro

Molecular and Cellular Biology ◽

10.1128/mcb.22.9.2974-2983.2002 ◽

2002 ◽

Vol 22 (9) ◽

pp. 2974-2983 ◽

Cited By ~ 58

Author(s):

Hiroshi Asahara ◽

Sophie Tartare-Deckert ◽

Takeya Nakagawa ◽

Tsuyoshi Ikehara ◽

Fumiko Hirose ◽

...

Keyword(s):

Transcriptional Activation ◽

Chromatin Assembly ◽

Micrococcal Nuclease ◽

Histone H2a ◽

Nucleosome Assembly ◽

Nucleosome Assembly Protein ◽

Nucleosome Assembly Protein 1 ◽

Core Histones ◽

Kix Domain

ABSTRACT In a yeast two-hybrid screen to identify proteins that bind to the KIX domain of the coactivator p300, we obtained cDNAs encoding nucleosome assembly protein 1 (NAP-1), a 60-kDa histone H2A-H2B shuttling protein that promotes histone deposition. p300 associates preferentially with the H2A-H2B-bound form of NAP-1 rather than with the unbound form of NAP-1. Formation of NAP-1-p300 complexes was found to increase during S phase, suggesting a potential role for p300 in chromatin assembly. In micrococcal nuclease and supercoiling assays, addition of p300 promoted efficient chromatin assembly in vitro in conjunction with NAP-1 and ATP-utilizing chromatin assembly and remodeling factor; this effect was dependent in part on the intrinsic histone acetyltransferase activity of p300. Surprisingly, NAP-1 potently inhibited acetylation of core histones by p300, suggesting that efficient assembly requires acetylation of either NAP-1 or p300 itself. As p300 acted cooperatively with NAP-1 in stimulating transcription from a chromatin template in vitro, our results suggest a dual role of NAP-1-p300 complexes in promoting chromatin assembly and transcriptional activation.

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Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.639587 ◽

2021 ◽

Vol 8 ◽

Author(s):

Marie-Laure Fogeron ◽

Lauriane Lecoq ◽

Laura Cole ◽

Matthias Harbers ◽

Anja Böckmann

Keyword(s):

Protein Synthesis ◽

Protein Expression ◽

Germ Cell ◽

Structural Biology ◽

Wheat Germ ◽

New Technologies ◽

Basic Research ◽

Free Protein ◽

Eukaryotic Proteins ◽

Cell Free Protein Synthesis

Cell-free protein synthesis (CFPS) systems are gaining more importance as universal tools for basic research, applied sciences, and product development with new technologies emerging for their application. Huge progress was made in the field of synthetic biology using CFPS to develop new proteins for technical applications and therapy. Out of the available CFPS systems, wheat germ cell-free protein synthesis (WG-CFPS) merges the highest yields with the use of a eukaryotic ribosome, making it an excellent approach for the synthesis of complex eukaryotic proteins including, for example, protein complexes and membrane proteins. Separating the translation reaction from other cellular processes, CFPS offers a flexible means to adapt translation reactions to protein needs. There is a large demand for such potent, easy-to-use, rapid protein expression systems, which are optimally serving protein requirements to drive biochemical and structural biology research. We summarize here a general workflow for a wheat germ system providing examples from the literature, as well as applications used for our own studies in structural biology. With this review, we want to highlight the tremendous potential of the rapidly evolving and highly versatile CFPS systems, making them more widely used as common tools to recombinantly prepare particularly challenging recombinant eukaryotic proteins.

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Gene expression in Acetabularia. I. Calibration of wheat germ cell-free translation system proteins as internal references for two-dimensional electrophoresis

Journal of Cell Science ◽

10.1242/jcs.58.1.23 ◽

1982 ◽

Vol 58 (1) ◽

pp. 23-33

Author(s):

R.L. Shoeman ◽

H.G. Schweiger

Keyword(s):

Germ Cell ◽

Isoelectric Point ◽

Wheat Germ ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

In Vitro Translation ◽

Translation System ◽

Two Dimensional ◽

Free Translation

Modification of existing two-dimensional techniques enables isoelectric focusing and sodium dodecyl sulphate polyacrylamide gel electrophoresis of complex mixtures of proteins to be completed within 8 h. The method was optimized to separate the protein components of a wheat germ cell-free translation system, providing a statistically proven resolution better than 0. 03 of a pH unit for the isoelectric point and 1000 for Mr. Fourteen of the more than 300 proteins separated were characterized with respect to Mr and isoelectric point relative to standard proteins under the same conditions. Stained wheat germ proteins thus serve as internal standards for analysis of in vitro translation products.

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