scholarly journals Accelerating the Production of Druggable Targets: Eukaryotic Cell-Free Systems Come into Focus

2019 ◽  
Vol 2 (2) ◽  
pp. 30 ◽  
Author(s):  
Thoring ◽  
Zemella ◽  
Wüstenhagen ◽  
Kubick
Keyword(s):  
Protein Expression ◽  
Posttranslational Modifications ◽  
Drug Targets ◽  
Protein Production ◽  
Production Systems ◽  
Low Cost ◽  
Functional Characterization ◽  
Eukaryotic Cell ◽  
Expression Systems ◽  
Mammalian Proteins

In the biopharmaceutical pipeline, protein expression systems are of high importance not only for the production of biotherapeutics but also for the discovery of novel drugs. The vast majority of drug targets are proteins, which need to be characterized and validated prior to the screening of potential hit components and molecules. A broad range of protein expression systems is currently available, mostly based on cellular organisms of prokaryotic and eukaryotic origin. Prokaryotic cell-free systems are often the system of choice for drug target protein production due to the simple generation of expression hosts and low cost of preparation. Limitations in the production of complex mammalian proteins appear due to inefficient protein folding and posttranslational modifications. Alternative protein production systems, so-called eukaryotic cell-free protein synthesis systems based on eukaryotic cell-lysates, close the gap between a fast protein generation system and a high quality of complex mammalian proteins. In this study, we show the production of druggable target proteins in eukaryotic cell-free systems. Functional characterization studies demonstrate the bioactivity of the proteins and underline the potential for eukaryotic cell-free systems to significantly improve drug development pipelines.

Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening

2014 ◽  
Vol 19 (7) ◽  
pp. 1000-1013 ◽  
Author(s):  
John W. Cuozzo ◽  
Holly H. Soutter
Keyword(s):  
Protein Expression ◽  
Small Molecule ◽  
Posttranslational Modifications ◽  
Expression Systems ◽  
Design Strategies ◽  
Protein Targets ◽  
Small Molecule Screening ◽  
Active Research ◽  
Small Molecule Modulators ◽  
And Function

Production of novel soluble and membrane-localized protein targets for functional and affinity-based screening has often been limited by the inability of traditional protein-expression systems to generate recombinant proteins that have properties similar to those of their endogenous counterparts. Such targets have often been labeled as challenging. Although biological validation of these challenging targets for specific disease areas may be strong, discovery of small-molecule modulators can be greatly delayed or completely halted due to target-expression issues. In this article, the limitations of traditional protein-expression systems will be discussed along with new systems designed to overcome these challenges. Recent work in this field has focused on two major areas for both soluble and membrane targets: construct-design strategies to improve expression levels and new hosts that can carry out the posttranslational modifications necessary for proper target folding and function. Another area of active research has been on the reconstitution of solubilized membrane targets for both structural analysis and screening. Finally, the potential impact of these new systems on the output of small-molecule screening campaigns will be discussed.

scholarly journals Decentralizing cell-free RNA sensing with the use of low-cost cell extracts

2021 ◽  
Author(s):  
Anibal Arce ◽  
Fernando Guzman ◽  
Chiara Gandini ◽  
Juan Puig ◽  
Tamara Matute ◽  
...  
Keyword(s):  
Protein Expression ◽  
Low Cost ◽  
Cold Chain ◽  
Expression Systems ◽  
Free Protein ◽  
Cell Extracts ◽  
Freeze Dried ◽  
Rna Sensors ◽  
Pure Cell ◽  
Pcr Products

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Here, we describe the implementation of RNA toehold switch-based sensors using E. coli cell lysate-based cell-free protein expression systems, which can be produced locally and reduce the cost of sensors by two orders of magnitude. We then demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize use of these lysates with a CRISPRi strategy to enhance the stability of linear DNAs, enabling the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toeholds sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

scholarly journals A Novel System for Rapid and Cost-Effective Production of Detection and Diagnostic Reagents of West Nile Virus in Plants

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Junyun He ◽  
Huafang Lai ◽  
Christopher Brock ◽  
Qiang Chen
Keyword(s):  
West Nile Virus ◽  
Posttranslational Modifications ◽  
Protein Production ◽  
Low Cost ◽  
Expression System ◽  
Cost Effective ◽  
West Nile ◽  
Economical System ◽  
High Scalability ◽  
Detection And Diagnosis

The threat of West Nile virus (WNV) epidemics necessitates the development of a technology platform that can produce reagents to support detection and diagnosis rapidly and inexpensively. A plant expression system is attractive for protein production due to its low-cost and high-scalability nature and its ability to make appropriate posttranslational modifications. Here, we investigated the feasibility of using plants to produce two WNV detection and diagnostic reagents to address the current cost and scalability issues. We demonstrated that WNV DIII antigen and E16 monoclonal antibody are rapidly produced at high levels in two plant species and are easily purified. Furthermore, they are effective in identifying WNV and in detecting human IgM response to WNV infection. E16 mAb does not cross-react with other flaviviruses, therefore, is valuable for improving diagnostic accuracy. This study provides a proof of principle for using plants as a robust and economical system to produce diagnostic reagents for arboviruses.

scholarly journals Preservation of protein expression systems at elevated temperatures for portable therapeutic production

2017 ◽  
Vol 14 (129) ◽  
pp. 20161039 ◽  
Author(s):  
David K. Karig ◽  
Seneca Bessling ◽  
Peter Thielen ◽  
Sherry Zhang ◽  
Joshua Wolfe
Keyword(s):  
Protein Expression ◽  
Elevated Temperatures ◽  
Low Cost ◽  
Efficient Production ◽  
Atmospheric Conditions ◽  
Expression Systems ◽  
Burn Wound ◽  
Long Term Stability ◽  
Wide Range ◽  
Application Potential

Many biotechnology capabilities are limited by stringent storage needs of reagents, largely prohibiting use outside of specialized laboratories. Focusing on a large class of protein-based biotechnology applications, we address this issue by developing a method for preserving cell-free protein expression systems for months above room temperature. Our approach realizes unprecedented long-term stability at elevated temperatures by leveraging the sugar alcohol trehalose, a simple, low-cost, open-air drying step, and strategic separation of reaction components during drying. The resulting preservation capacity enables efficient production of a wide range of on-demand proteins under adverse conditions, for instance during emergency outbreaks or in remote locations. To demonstrate application potential, we use cell-free reagents subjected to months of exposure at 37°C and atmospheric conditions to produce sufficient concentrations of a pyocin protein to kill Pseudomonas aeruginosa , a troublesome pathogen for traumatic and burn wound injuries. Our work makes possible new biotechnology applications that demand ruggedness and scalability.

Causes of gear pump fails and methods for their elimination

2020 ◽  
pp. 98-107
Author(s):  
Vitaliy A. Zuyevskiy ◽  
Daniil O. Klimyuk ◽  
Ivan A. Shemberev
Keyword(s):  
Adhesion Strength ◽  
Production Systems ◽  
Low Cost ◽  
High Strength ◽  
Strength Properties ◽  
Research Purpose ◽  
Working Fluid ◽  
Gear Pump ◽  
Repair Service ◽  
Recovery Method

Gear pumps are an important element of many production systems and their replacement in case of failure can be quite expensive, so it is important to have a modern and well-tuned technology for their recovery. There are many methods for restoring the pump's performance, depending on the reason that led to its failure. (Research purpose) The research purpose is in determining what causes most often lead to loss of pump performance, and developing a recovery method that provides the greatest post-repair service life of the pump and low cost of repair. (Materials and methods) Authors took into account that the applied coatings must have sufficient adhesion strength and resistance to mechanical, thermal and corrosion loads during operation. It was found that most often significant leaks of the working fluid, leading to failure, occur due to an increase in the gap between the inner surface of the housing and the gears due to active wear of the housing wells. Authors determined that the method of electric spark treatment of worn-out housing wells is best suited to perform the task (a large post-repair resource and low costs). (Results and discussion) It was found by laboratory studies of the adhesion strength of electric spark coatings with various electrodes that the best transfer of the material to the substrate is provided by bronze electrodes BrMKts3-1. It was noted that the coatings applied using the BrMKts3-1 electrode have high strength properties. (Conclusions) Research conducted in the center for collective use "Nano-Center" VIM confirmed the possibility of effective recovery of the gear pump by electric spark treatment.

scholarly journals Characterization of synthetic riboswitch in cell-free protein expression systems

RNA Biology ◽  
2021 ◽  
pp. 1-12
Author(s):  
Yaroslav Chushak ◽  
Svetlana Harbaugh ◽  
Kathryn Zimlich ◽  
Bryan Alfred ◽  
Jorge Chávez ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Systems ◽  
Free Protein

scholarly journals Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Jennifer A. Schmidt ◽  
Lubna V. Richter ◽  
Lisa A. Condoluci ◽  
Beth A. Ahner
Keyword(s):  
Protein Expression ◽  
Protein Production ◽  
Biomass Accumulation ◽  
Cellulase Production ◽  
Protein Yield ◽  
Total Soluble Protein ◽  
Foreign Protein ◽  
Active Components ◽  
Dwarf Phenotype ◽  
Mutant Phenotypes

Abstract Background The global demand for functional proteins is extensive, diverse, and constantly increasing. Medicine, agriculture, and industrial manufacturing all rely on high-quality proteins as major active components or process additives. Historically, these demands have been met by microbial bioreactors that are expensive to operate and maintain, prone to contamination, and relatively inflexible to changing market demands. Well-established crop cultivation techniques coupled with new advancements in genetic engineering may offer a cheaper and more versatile protein production platform. Chloroplast-engineered plants, like tobacco, have the potential to produce large quantities of high-value proteins, but often result in engineered plants with mutant phenotypes. This technology needs to be fine-tuned for commercial applications to maximize target protein yield while maintaining robust plant growth. Results Here, we show that a previously developed Nicotiana tabacum line, TetC-cel6A, can produce an industrial cellulase at levels of up to 28% of total soluble protein (TSP) with a slight dwarf phenotype but no loss in biomass. In seedlings, the dwarf phenotype is recovered by exogenous application of gibberellic acid. We also demonstrate that accumulating foreign protein represents an added burden to the plants’ metabolism that can make them more sensitive to limiting growth conditions such as low nitrogen. The biomass of nitrogen-limited TetC-cel6A plants was found to be as much as 40% lower than wildtype (WT) tobacco, although heterologous cellulase production was not greatly reduced compared to well-fertilized TetC-cel6A plants. Furthermore, cultivation at elevated carbon dioxide (1600 ppm CO2) restored biomass accumulation in TetC-cel6A plants to that of WT, while also increasing total heterologous protein yield (mg Cel6A plant−1) by 50–70%. Conclusions The work reported here demonstrates that well-fertilized tobacco plants have a substantial degree of flexibility in protein metabolism and can accommodate considerable levels of some recombinant proteins without exhibiting deleterious mutant phenotypes. Furthermore, we show that the alterations to protein expression triggered by growth at elevated CO2 can help rebalance endogenous protein expression and/or increase foreign protein production in chloroplast-engineered tobacco.

Concept to Support the Flexibility of Manufacturing System through Reconfigurable Structure Based on Modular Design

2015 ◽  
Vol 816 ◽  
pp. 536-546
Author(s):  
Vladimír Rudy ◽  
Andrea Lešková
Keyword(s):  
Flexible Manufacturing ◽  
Manufacturing Systems ◽  
Structural Changes ◽  
Modular Design ◽  
Production Systems ◽  
Low Cost ◽  
Modular Construction ◽  
Production Platform ◽  
Flexible Adaptation ◽  
Reconfigurable Structure

This article deals about the challenges of structural changes in manufacturing conditions. The objective of this paper is to present the modular workstations concept based on miniaturization and re-configurability trends. The article is aimed at problems of designing of production systems with a modular construction structure. The modular structure allows an individual and flexible adaptation to varying requirements but also the realization of low-cost solutions for creation of new or modernized production base. The goal is to present the example of modular workstations solutions that correspond with new designing approach. The specification of basics principles, which should help to designing flexible manufacturing systems, discussed in this paper are: modularity; integrability; convertibility; diagnosability; customization. The theoretical part provides an overview of fundamental design principles in manufacturing structures. In the first part of this article are discussed the specification of basic flexibility types in production system and the main impacts influencing design of manufacturing structures. The closing section of the article provides the specification of example solution of adjustable production platform with modular frame (called desktop factory).

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