Article
Multidisciplinary Sciences
Thapakorn Jaroentomeechai, Yong Hyun Kwon, Yiwen Liu, Olivia Young, Ruchika Bhawal, Joshua D. Wilson, Mingji Li, Digantkumar G. Chapla, Kelley W. Moremen, Michael C. Jewett, Dario Mizrachi, Matthew P. DeLisa
Summary: This study describes a strategy to convert membrane-bound glycosyltransferases into water soluble biocatalysts, which allows for easier expression and structural remodeling of glycans. The approach was successfully used to produce 98 difficult-to-express GTs, predominantly of human origin, and demonstrated its universality and versatility.
NATURE COMMUNICATIONS
(2022)
Article
Biotechnology & Applied Microbiology
Xiaomei Lin, Caijin Zhou, Ting Wang, Xiaoting Huang, Junxin Chen, Zhixia Li, Jisong Zhang, Yuan Lu
Summary: This study found that carbon dioxide can directly affect protein synthesis and increase protein yield. Under high oxygen conditions, the combined effect of carbon dioxide and oxygen can further enhance protein expression. In addition, carbon dioxide can promote protein synthesis by improving energy metabolism and redox balance.
SYNTHETIC AND SYSTEMS BIOTECHNOLOGY
(2022)
Article
Biochemical Research Methods
Christopher W. Koo, Jasmine M. Hershewe, Michael C. Jewett, Amy C. Rosenzweig
Summary: The study developed a cell-free protein synthesis system based on Escherichia coli lysate, which can be used to express particulate methane monooxygenase (pMMO) in vitro in the presence of nanodiscs. The findings demonstrate that the pMMO complex can form without the need for exogenous translocon machinery or chaperones.
ACS SYNTHETIC BIOLOGY
(2022)
Article
Biochemical Research Methods
Daisuke Inoue, Keisuke Ohashi, Taichi E. Takasuka, Akira Kakugo
Summary: In this study, we successfully synthesized and edited functional kinesins using a wheat germ cell-free protein synthesis system. The synthesized kinesins propelled microtubules on a kinesin-coated substrate and showed higher binding affinity with microtubules compared to E. coli-produced kinesins. We also successfully incorporated affinity tags into the kinesins using PCR. Our method will accelerate the study of biomolecular motor systems and promote their wider use in various nanotechnology applications.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Zhixia Li, Yuting Li, Xiaomei Lin, Yuntao Cui, Ting Wang, Jian Dong, Yuan Lu
Summary: Protein-based biomaterials, with the characteristics of stability and biocompatibility, can be synthesized and assembled in situ using cell-free protein synthesis (CFPS) technology, which shortens the synthesis time and increases flexibility.
BIORESOURCES AND BIOPROCESSING
(2022)
Review
Biochemistry & Molecular Biology
Ke Yue, Junyu Chen, Yingqiu Li, Lei Kai
Summary: The rapid development of synthetic biology has enabled revolutionary improvements in biotechnology. Cell-free protein synthesis (CFPS) has been instrumental in further advancing synthetic biology through its ability to directly manipulate the Central Dogma with flexibility and rapid feedback. This mini-review summarizes recent achievements and applications of CFPS technique in various synthetic biology projects, including minimal cell assembly, metabolic engineering, recombinant protein production for therapeutics, and biosensor development for in vitro diagnostics. It also outlines current challenges and future perspectives in developing a generalized cell-free synthetic biology.
COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL
(2023)
Article
Biotechnology & Applied Microbiology
You Jin Lee, Soojin Lee, Dong-Myung Kim
Summary: Gene expression can efficiently amplify DNA information into protein molecules. Cell-free protein synthesis (CFPS) offers a solution to analyze biomolecules in vitro without the problems associated with whole cell-based approaches. In this study, the authors developed a method to analyze indole using enzymatic conversion and amino acid-dependent CFPS, successfully estimating the indole concentration.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2022)
Article
Chemistry, Analytical
Hiroki Nakai, Kinuka Isshiki, Masato Hattori, Hiromasa Maehira, Tatsumi Yamaguchi, Keiko Masuda, Yoshihiro Shimizu, Takayoshi Watanabe, Takahiro Hohsaka, Wataru Shihoya, Osamu Nureki, Yasuhiko Kato, Hajime Watanabe, Tomoaki Matsuura
Summary: This study developed a high-throughput screening method for GPCRs using an in vitro transcription-translation (IVTT) system. Functional ETBR and ETAR were synthesized in nanodiscs using the IVTT system and subjected to ribosome display. POPG was found to support the folding of ETBR into its functional form, suggesting the possibility of high-throughput screening and directed evolution of GPCRs in vitro.
ANALYTICAL CHEMISTRY
(2022)
Article
Chemistry, Organic
Alessio M. Caramiello, Maria Cristina Bellucci, Gaetano Cristina, Carlo Castellano, Fiorella Meneghetti, Matteo Mori, Francesco Secundo, Fiorenza Viani, Alessandro Sacchetti, Alessandro Volonterio
Summary: This study presents the synthesis of a collection of enantiomerically pure, systematically substituted hydantoins as structural privileged universal mimetic scaffolds. The synthesis relies on a selective condensation/cyclization process between isocyanates and aspartic acid diesters, followed by hydrolysis/coupling reactions with amines. Conformational studies showed that the hydantoin-based peptidomimetics are capable of projecting their substituents in positions superimposable to common protein secondary structures.
JOURNAL OF ORGANIC CHEMISTRY
(2022)
Article
Biotechnology & Applied Microbiology
Ruben Magnus Walter, Anne Zemella, Marina Schramm, Jan Kiebist, Stefan Kubick
Summary: Unspecific peroxygenases (UPOs) are valuable biocatalysts, but there is currently no universal expression system. This study established an insect-based cell-free protein synthesis (CFPS) platform and successfully synthesized a novel UPO, demonstrating the potential of CFPS for UPO discovery and evaluation.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Yanan Wang, Ting Wang, Xinjie Chen, Yuan Lu
Summary: A cell-free protein synthesis (CFPS) system based on Pichia pastoris yeast was constructed, providing a low-cost and time-efficient platform for protein research and synthesis. The combination of IRES and Kozak sequences revealed that cricket paralysis virus (CRPV) was the best translation initiation element among 14 different IRESs. Optimization of system components and reaction environment led to increased protein yield. This P. pastoris CFPS system extends the eukaryotic CFPS platform and enables fast prototyping design and functional protein synthesis.
BIORESOURCES AND BIOPROCESSING
(2023)
Article
Multidisciplinary Sciences
Jasmine M. Hershewe, Katherine F. Warfel, Shaelyn M. Iyer, Justin A. Peruzzi, Claretta J. Sullivan, Eric W. Roth, Matthew P. DeLisa, Neha P. Kamat, Michael C. Jewett
Summary: In this study, native membrane vesicles in E. coli extracts were characterized for enhanced glycoengineering. Enriching vesicles in the extracts led to increased concentrations of heterologous membrane protein cargo. These methods are expected to facilitate glycoprotein production and enable new CFE systems with membrane-associated activities.
NATURE COMMUNICATIONS
(2021)
Article
Biotechnology & Applied Microbiology
Nan Jiang, Xuanwei Ding, Yuan Lu
Summary: The cell-free protein synthesis system offers advantages of flexible control over reaction environment and ability to synthesize proteins that are difficult to make in vivo, but expression efficiency remains a challenge. This study demonstrates that in the optimized cell-free system, a concentration of 15mM Mg2+ can better synthesize proteins.
BIOCHEMICAL ENGINEERING JOURNAL
(2021)
Review
Biochemical Research Methods
Hye Jin Lim, Dong-Myung Kim
Summary: Cell-free synthesis is an efficient method that decouples biosynthetic reactions from cellular physiology, enabling fast and flexible production of biomolecules, industrial chemicals, and biofuels. It is considered a promising alternative to microbial fermentation processes.
CURRENT OPINION IN BIOTECHNOLOGY
(2022)
Review
Biotechnology & Applied Microbiology
Jiaojiao Li, Mengtong Tang, Hao Qi
Summary: Manipulating tRNAs can simplify genetic codons in a cell-free protein synthesis system and offer more flexibility. This article provides an overview of tRNA complement protein synthesis system construction and strategies for removing tRNAs from cell lysates and synthesizing tRNAs in vivo/vitro. It also highlights the trend towards reducing codon redundancy by manipulating tRNAs in different proteins. The tRNA complement protein synthesis system has the potential to facilitate the construction of minimal cells and expand the biomedical application scope of synthetic biology.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2022)