Repressor‐Like On‐Off Regulation of Protein Expression by the DNA‐Binding Transcription Activator‐Like Effector in T7 Promoter‐Based Cell‐Free Protein Synthesis

ChemBioChem ◽  
2020 ◽  
Author(s):  
Masafumi Sakono ◽  
Ryoto Hayakawa
Keyword(s):  
Protein Synthesis ◽  
Dna Binding ◽  
Protein Expression ◽  
Transcription Activator ◽  
T7 Promoter ◽  
Free Protein ◽  
Cell Free Protein Synthesis

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

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.

scholarly journals Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression

Biochemistry ◽  
2016 ◽  
Vol 55 (30) ◽  
pp. 4212-4219 ◽  
Author(s):  
Paul J. Focke ◽  
Christopher Hein ◽  
Beate Hoffmann ◽  
Kimberly Matulef ◽  
Frank Bernhard ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Membrane Protein ◽  
Protein Expression ◽  
Free Protein ◽  
Membrane Protein Expression ◽  
Cell Free Protein Synthesis

scholarly journals Cell-Free Expression of Protein Kinase a for Rapid Activity Assays

2010 ◽  
Vol 5 ◽  
pp. ACI.S4732 ◽  
Author(s):  
Donna M. Leippe ◽  
Kate Qin Zhao ◽  
Kevin Hsiao ◽  
Michael R. Slater
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Expression ◽  
Magnetic Beads ◽  
Protein Analysis ◽  
Functional Protein ◽  
Free Protein ◽  
Protein Expression And Purification ◽  
Cell Free Protein Synthesis

Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag® fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.

scholarly journals A User’s Guide to Cell-Free Protein Synthesis

2019 ◽  
Vol 2 (1) ◽  
pp. 24 ◽  
Author(s):  
Nicole E. Gregorio ◽  
Max Z. Levine ◽  
Javin P. Oza
Keyword(s):  
Protein Synthesis ◽  
Protein Expression ◽  
Living Cells ◽  
Development Education ◽  
Free Protein ◽  
Therapeutic Development ◽  
A Cell ◽  
New Applications ◽  
Cell Free Protein Synthesis

Cell-free protein synthesis (CFPS) is a platform technology that provides new opportunities for protein expression, metabolic engineering, therapeutic development, education, and more. The advantages of CFPS over in vivo protein expression include its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest. Over the last 60 years, the CFPS platform has grown and diversified greatly, and it continues to evolve today. Both new applications and new types of extracts based on a variety of organisms are current areas of development. However, new users interested in CFPS may find it challenging to implement a cell-free platform in their laboratory due to the technical and functional considerations involved in choosing and executing a platform that best suits their needs. Here we hope to reduce this barrier to implementing CFPS by clarifying the similarities and differences amongst cell-free platforms, highlighting the various applications that have been accomplished in each of them, and detailing the main methodological and instrumental requirement for their preparation. Additionally, this review will help to contextualize the landscape of work that has been done using CFPS and showcase the diversity of applications that it enables.

scholarly journals A Method for Cost-Effective and Rapid Characterization of Engineered T7-based Transcription Factors by Cell-Free Protein Synthesis Reveals Insights into the Regulation of T7 RNA Polymerase-Driven Expression

2019 ◽  
Author(s):  
John B. McManus ◽  
Richard M. Murray ◽  
Peter A. Emanuel ◽  
Matthew W. Lux
Keyword(s):  
Protein Synthesis ◽  
Transcription Factors ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Circuit Design ◽  
Cost Effective ◽  
Gene Circuit ◽  
Free Protein ◽  
Cost Effective Method ◽  
Cell Free Protein Synthesis

AbstractThe T7 bacteriophage RNA polymerase (T7 RNAP) serves as a model for understanding RNA synthesis, as a tool for protein expression, and as an actuator for synthetic gene circuit design in bacterial cells and cell-free extract. T7 RNAP is an attractive tool for orthogonal protein expression in bacteria owing to its compact single subunit structure and orthogonal promoter specificity. Understanding the mechanisms underlying T7 RNAP regulation is important to the design of engineered T7-based transcription factors, which can be used in gene circuit design. To explore regulatory mechanisms for T7 RNAP-driven expression, we developed a rapid and cost-effective method to characterize engineered T7-based transcription factors using cell-free protein synthesis and an acoustic liquid handler. Using this method, we investigated the effects of the tetracycline operator’s proximity to the T7 promoter on the regulation of T7 RNAP-driven expression. Our results reveal a mechanism for regulation that functions by interfering with the transition of T7 RNAP from initiation to elongation and validates the use of the method described here to engineer future T7-based transcription factors.HighlightsDevelopment of a rapid and cost-effective method for screening synthetic promoters.Insights into the regulation of engineered T7-based transcription factors and T7 RNAP enzyme kinetics.Validation of this method by comparison with the T7 RNAP kinetic model.

scholarly journals Cell-free protein synthesis of norovirus virus-like particles

RSC Advances ◽  
2017 ◽  
Vol 7 (46) ◽  
pp. 28837-28840 ◽  
Author(s):  
Jiayuan Sheng ◽  
Shaohua Lei ◽  
Lijuan Yuan ◽  
Xueyang Feng
Keyword(s):  
Protein Synthesis ◽  
Protein Expression ◽  
Free Protein ◽  
Virus Like Particles ◽  
Cell Free Protein Synthesis

Cell-free protein expression of norovirus virus-like-particles.

scholarly journals Cell-free protein synthesis and in situ immobilization of deGFP-MatB in polymer microgels for malonate-to-malonyl CoA conversion

RSC Advances ◽  
2020 ◽  
Vol 10 (66) ◽  
pp. 40588-40596
Author(s):  
Tony Köhler ◽  
Thomas Heida ◽  
Sandra Hoefgen ◽  
Niclas Weigel ◽  
Vito Valiante ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Genetic Information ◽  
In Situ Immobilization ◽  
Bottom Up ◽  
Free Protein ◽  
Malonyl Coa ◽  
Cell Free Protein Synthesis

We describe a bottom-up approach towards functional enzymes utilizing microgels as carriers for genetic information that enable cell-free protein synthesis, in situ immobilization, and utilization of functional deGFP-MatB.

Sign in / Sign up

Export Citation Format

Share Document