Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Abstract

Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab‐on‐chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell‐free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell‐cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell‐free protein synthesis (CFPS) formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed.

Potential Applications of Advanced Nano/Hydrogels in Biomedicine: Static, Dynamic, Multi‐Stage, and Bioinspired

“…as differentiation, as discussed in our previous review. [15] A DNA hydrogel with the ability to carry small interfering RNA (siRNA) used to silence protein expression was fabricated by Song et.al. [38] In this study, the gene that encoded the siRNA was located on a plasmid, which itself was part of the hydrogel scaffold, hence it was termed RNAi-inhibiting gel or I-gel.…”

“…Non‐biodegradable hydrogels can act as a mimicking structure that could be a replacement for living cells, for instance for in situ cell‐free gene/protein expression or to model cell–cell communications to understand cellular processes such as differentiation, as discussed in our previous review. [ 15 ] A DNA hydrogel with the ability to carry small interfering RNA (siRNA) used to silence protein expression was fabricated by Song et.al. [ 38 ] In this study, the gene that encoded the siRNA was located on a plasmid, which itself was part of the hydrogel scaffold, hence it was termed RNAi‐inhibiting gel or I‐gel.…”

“…[ 6 ] Hydrogels can be used as microcompartments for in situ cell‐free protein expression from specific genes in order to study gene therapy and cancer therapy applications. [ 15 ] Because hydrogels can be loaded with various types of cargo, ranging from small molecules to macromolecular theranostics, or even living cells, they can be designed for sustained and controlled release of the cargo at the site of interest. They can be applied in immunotherapy, where local administration of antibody‐loaded hydrogels as bioimplants or as sprays could be used to modulate immune responses, eliciting a systemic anti‐tumor immune response after surgery to prevent cancer recurrence.…”

Plant transporters involved in combating boron toxicity: beyond 3D structures

“…One of the progressive membrane protein production approaches includes high-yield and high-titer cellular bioproduction systems [ 71 ] in synthetic biology cell-free protein synthesis open formats [ 72 , 73 ] harboured in minimal cells and microfluidic devices [ 74 ]. These robust micro-reactors and micro-compartments offer novel platforms and integrate tasks in parallel, where the emphasis is on a low cost and convenience (e.g.…”

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

“…The diverse features of CFPS systems promoted also their recent use in teaching (Stark et al, 2019), protein engineering (Kido et al, 2020), and synthetic biology (Tinafar et al, 2019), which holds great promises for studies on genetic networks or rapid prototyping (Karim et al, 2020) in metabolic engineering (Perez et al, 2016) as well as future drug development (Dondapati et al, 2020). Moreover, the in vitro reaction format of CFPS systems allows for full automation, miniaturization (Ayoubi-Joshaghani et al, 2020), and working with large sample numbers (Zhu et al, 2015). This advantage has been utilized in large-scale screening experiments (Khnouf et al, 2010;Kim et al, 2015), searches for malaria vaccine candidates (Kanoi et al, 2017;Morita et al, 2017;Kanoi et al, 2020), identifying interactions between E3 ligases and their substrates (Takahashi et al, 2016), building a protein array holding human Deubiquitinating Enzymes (DUBs) (Takahashi et al, 2020), or the development of protein array platforms (Romanov et al, 2014;Zarate and Galbraith, 2014;Morishita et al, 2019).…”