Cell‐Free Protein Synthesis in Bifunctional Hyaluronan Microgels: A Strategy for In Situ Immobilization and Purification of His‐Tagged Proteins

Abstract

The front cover artwork is provided by the Thiele group at Leibniz IPF Dresden (Germany). The image shows a microgel hosting cell‐free synthesis of green fluorescent protein. Despite its open, membrane‐free nature, the multifunctional microgel is also able to trap proteins inside its polymer matrix. Read the full text of the Article at 10.1002/syst.201900058.

Cell-free protein synthesis andin situimmobilization of deGFP-MatB in polymer microgels for malonate-to-malonyl CoA conversion

“…12,13,17,18,39 However, to efficiently couple the template DNA to our hydrogel matrix, linear PCR products were used for functionalization. 7,10 Screening for optimal DNA concentration, depicted in Fig. 4, indicated an optimum of 5 nM with a yield of 62.2 AE 10.9 mg mL À1 , whereas 10 nM gave a rather similar yield of 52.4 AE 9.9 mg mL À1 , for deGFP-MatB.…”

“…Due to its large variability, cell-free protein synthesis (CFPS) is a key tool in synthetic biology, which has been successfully combined with cell-sized entities, such as vesicles, 1 emulsions, 2,3 microcapsules 4,5 or microgels. [6][7][8][9][10] By tuning the composition regarding ribosomes, t-RNA-acyltransferases, transcription as well as translation factors, among others 11 -easily extracted from Escherichia coli (E. coli) by different techniques, e.g., sonication 12,13 or bead-beating 14 -CFPS systems with tailor-made protein synthesis kinetics, 15 reaction duration, 16 and reaction medium crowding 2 are prepared. Additionally, these components can also be expressed as recombinant proteins to simplify CFPS preparation.…”

“…In these approaches, DNA, which is coding for the protein of interest, is either diffusing through the reaction mixture [1][2][3][4][5]8 or coupled to a matrix such as polymer microgels. 6,7,9,10 Microgels consist of a porous polymer matrix swollen in a solventin this context, the aqueous CFPS reaction mixture, for instance. In contrast to more established articial microenvironments, e.g., vesicles, microgels are not surrounded by a membrane that enables controlled and switchable mass transfer.…”

“…24 To design biocompatible microgels in a bio-orthogonal fashion, natural building blocks like polysaccharides are well-suited, as modications of their backbone for cross-linking and functionalization have been thoroughly studied to come up with both, physicochemically and mechanically dened polysaccharide microgels for cell-free biotechnology and cell biology. 6,[25][26][27] Along these lines, hyaluronic acid (HA) allows for introducing a wide range of modications, e.g., tyramine, methacrylates, thiols, vinyl esters or (methyl)furan groups, 7,10,[25][26][27] demonstrating the versatility of this material basis for microgel formation. By combining these modications, multi-functional HA-based microgels are obtained allowing for coupling both DNA as genetic information in CFPS, and for further functionalizing the hydrogel matrix with anchoring groups, e.g., with selective binding motives, to control diffusion of as-formed protein out of the porous polymer matrix.…”

“…20,[29][30][31] The application of enzymes is conventionally based on heterologous expression in an organism such as E. coli, being a time-consuming procedure that takes days, 21,32 while CFPS systems can produce proteins within a few hours. 13 The combination of CFPS with a hydrogel-based approach was yet more oen used to synthesize well-known proteins such as deGFP 7,10 or mVenus. 6 However, the aim should be to produce functional enzymes, for instance, capable of synthesizing building blocks for further biosynthetic compound design.…”