Expanding One-Pot Cell-Free Protein Synthesis and Immobilization for On-Demand Manufacturing of Biomaterials

Abstract

Fabrication of protein-based biomaterials is an arduous and time-consuming procedure with multiple steps. In this work, we describe a portable toolkit that integrates both cell-free protein synthesis (CFPS) and protein immobilization in one pot just by mixing DNA, solid materials, and a CFPS system. We have constructed a modular set of plasmids that fuse the N-terminus of superfolded green fluorescent protein (sGFP) with different peptide tags (poly(6X)Cys, poly(6X)His, and poly(6X)Lys), which drive the immobilization of the protein on the tailored material (agarose beads with different functionalities, gold nanorods, and silica nanoparticles). This system also enables the incorporation of azide-based amino acids into the nascent protein for its selective immobilization through copper-free click reactions. Finally, this technology has been expanded to the synthesis and immobilization of enzymes and antibody-binding proteins for the fabrication of functional biomaterials. This synthetic biological platform has emerged as a versatile tool for on-demand fabrication of therapeutic, diagnostic, and sensing biomaterials.

The Genetic Code Kit: An Open-Source Cell-Free Platform for Biochemical and Biotechnology Education

“…Advancements in the CFPS platform over the last few decades have enabled a multitude of novel applications in biotechnology, including rapid prototyping for engineering biological systems and easy-to-use point of care diagnostics and biosensors (Pardee et al, 2016;Salehi et al, 2017;Benítez-Mateos et al, 2018;Bundy et al, 2018;Dopp and Reuel, 2018;Takahashi et al, 2018;Gräwe et al, 2019;Gregorio et al, 2019;Silverman et al, 2019;Jung et al, 2020). CFPS generally relies on a cell-extract containing the cellular machinery that supports transcription and translation in vitro and is supplemented with additional reagents that provide the necessary energy and precursors.…”

Advances in Cell‐Free Biosensors: Principle, Mechanism, and Applications

“…With further evolution of cell‐free biosensors, besides environmental monitoring and human health diagnosis, they will increasingly expand their applications to food testing, classroom education, and others, making them more practical and commercial. [ 52,97,116,117 ]…”

Construction and optimization of microbial cell factories for sustainable production of bioactive dammarenediol-II glucosides

“…Apart from enzyme catalysis, 7,8 metabolic engineering as one of the green and sustainable biotechnologies has been extensively applied to not only refactor metabolic pathways of natural products in microbes for their heterologous synthesis but also generate unnatural products and even new compounds by artificially designing their metabolic pathways. 9 The development of metabolic engineering allows the construction of more desirable microbial cell factories for the production of the target products.…”