Expanding one-pot cell-free protein synthesis and immobilization for on-demand manufacturing of biomaterials

Fabrication of protein-based biomaterials is an arduous and time-consuming procedure with multiple steps. In this work, we describe a portable tool-kit 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 construct a modular set of plasmids that fuse the N-terminus of superfolded Green Fluorescent Protein (sGFP) with different peptide-tags (poly-Cys, poly-His and poly-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 aminoacids into the nascent protein for its selective immoblization 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 emerges as a versatile tool for the on-demand fabrication of therapeutic, diagnostic and sensing biomaterials.We want to acknowledge COST action CM103-System biocatalysis, IKERBASQUE, Basque foundation of Science and ARAID, Aragon I+D foundation for funding to Dr. F. López-Gallego and to Spanish Minister MINECO (BIO2014-61838-EXP) for funding A. I. Benítez-Mateos. We want to thank Dr. C. Araya-Callís and Dr. R. P. Richter for the kind donation of plasmid encoding His-ProA protein. Likewise, we also thank Dr. A. Basso from Purolite Ltd for kindly donating us some samples of Purolite® resins