cell-free-protein-synthesis-system-for-building-synthetic-cells

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

Jiangsu Normal University

The quest for a minimal cell not only sheds light on the fundamental principles of life but also brings great advances in related applied fields such as general biotechnology. Minimal cell projects came from the study of a plausible route to the origin of life. Later on, research extended and also referred to the construction of artificial cells, or even more broadly, as in vitro synthetic biology. The cell-free protein synthesis (CFPS) techniques harness the central cellular activity of transcription/translation in an open environment, providing the framework for multiple cellular processes assembling. Therefore, CFPS systems have become the first choice in the construction of the minimal cell. In this review, we focus on the recent advances in the quantitative analysis of CFPS and on its advantage for addressing the bottom-up assembly of a minimal cell and illustrate the importance of systemic chassis behavior, such as stochasticity under a compartmentalized micro-environment.

Cell-Free Protein Synthesis: Chassis toward the Minimal Cell.

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from  and  using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and PIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an  Based Cell-Free Protein Synthesis System.

Aquaporins are essential water channel proteins found in all kingdoms of life. Although the water permeability of aquaporins has been well characterized, sample preparation for aquaporin water permeability assays remains challenging and time-consuming. Besides the difficulty in overexpressing membrane proteins in a cell-based expression system, the unique requirement for homogeneity in aquaporin proteoliposome sample preparations for water transport assays further increases the complexity. In this study, a complementary Cell-free Protein Synthesis (CFPS) method is described in detail, providing three different strategies for the preparation of aquaporin proteoliposome samples. Aquaporin can be produced either as a pellet fraction and then resolubilized, or co-translationally as a detergent-soluble fraction. Furthermore, aquaporin can be directly incorporated into liposomes, which was included in the CFPS reactions. Although proteoliposomes tend to fuse during the incubation of the CFPS reactions, an additional treatment of the fused samples with detergent, followed by a detergent removal step, can re-form homogenously sized proteoliposomes suitable for functional analysis. Using this method, we successfully characterized aquaporins from both prokaryotic and eukaryotic organisms. In particular, in the presence of liposomes, the developed CFPS expression system is a fast and convenient method for sample preparation for the functional analysis of aquaporins.

Functional Analysis of Aquaporin Water Permeability Using an -Based Cell-Free Protein Synthesis System.

The rapid development of synthetic biology has enabled the production of compounds with revolutionary improvements in biotechnology. DNA manipulation tools have expedited the engineering of cellular systems for this purpose. Nonetheless, the inherent constraints of cellular systems persist, imposing an upper limit on mass and energy conversion efficiencies. Cell-free protein synthesis (CFPS) has demonstrated its potential to overcome these inherent constraints and has been instrumental in the further advancement of synthetic biology. Via the removal of the cell membranes and redundant parts of cells, CFPS has provided flexibility in directly dissecting and manipulating the Central Dogma with rapid feedback. This mini-review summarizes recent achievements of the CFPS technique and its application to a wide range of synthetic biology projects, such as minimal cell assembly, metabolic engineering, and recombinant protein production for therapeutics, as well as biosensor development for in vitro diagnostics. In addition, current challenges and future perspectives in developing a generalized cell-free synthetic biology are outlined.

Advancing synthetic biology through cell-free protein synthesis.

Despite the physiological significance of effective CO diffusion across biological membranes, the underlying mechanism behind this process is not yet resolved. Particularly debatable is the existence of CO-permeable aquaporins. The lipophilic characteristic of CO should, according to Overton's rule, result in a rapid flux across lipid bilayers. However, experimental evidence of limited membrane permeability poses a challenge to this idea of free diffusion. In this review, we summarized recent progress with regard to CO diffusion, and discussed the physiological effects of altered aquaporin expression, the molecular mechanisms of CO transport via aquaporins, and the function of sterols and other membrane proteins in CO permeability. In addition, we highlight the existing limits in measuring CO permeability and end up with perspectives on resolving such argument either by determining the atomic resolution structure of CO permeable aquaporins or by developing new methods for measuring permeability.

Aquaporins and CO diffusion across biological membrane.

Technical advances in biotechnology have greatly accelerated the development of bottom-up synthetic biology. Unlike top-down approaches, bottom-up synthetic biology focuses on the construction of a minimal cell from scratch and the application of these principles to solve challenges. Cell-free protein synthesis (CFPS) systems provide minimal machinery for transcription and translation, from either a fractionated cell lysate or individual purified protein elements, thus speeding up the development of synthetic cell projects. In this review, we trace the history of the cell-free technique back to the first in vitro fermentation experiment using yeast cell lysate. Furthermore, we summarized progresses of individual cell mimicry modules, such as compartmentalization, gene expression regulation, energy regeneration and metabolism, growth and division, communication, and motility. Finally, current challenges and future perspectives on the field are outlined.

Article	Year
Cell-Free Protein Synthesis: Chassis toward the Minimal Cell. Cells\| PubMed ID: 30959805	2019
Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an Based Cell-Free Protein Synthesis System. Cells\| PubMed ID: 31717877	2019
Functional Analysis of Aquaporin Water Permeability Using an -Based Cell-Free Protein Synthesis System. Frontiers in bioengineering and biotechnology\| PubMed ID: 32974321	2020
Advancing synthetic biology through cell-free protein synthesis. Computational and structural biotechnology journal\| PubMed ID: 37216017	2023
Aquaporins and CO diffusion across biological membrane. Frontiers in physiology\| PubMed ID: 37383148	2023
Bottom-Up Synthetic Biology Using Cell-Free Protein Synthesis. Advances in biochemical engineering/biotechnology\| PubMed ID: 37526707	2023

Ke Yue

Biography

JoVE Journal Publications

Other Publications