Liquid marble (LM), a self-standing micro-scale aqueous droplet, emerges as a micro-bioreactor in biological applications. Herein, the potential of LM as media for cell-free synthesis and simultaneous immobilization of recombinant proteins is explored. Initially, formation of hydrogel marble (HM) by using an enzymatic disulfide-based hydrogelation technique is confirmed by incorporating three components, horseradish peroxidase (HRP), a tetra-thiolated poly(ethylene glycol) derivative, and glycyl-L-tyrosine, in LM. The compatibility of the enzymatic hydrogelation with cell-free protein synthesis in LM is then validated. Although the hydrogelation reduces the level of protein synthesis in LM when compared with that in a test tube, the biosynthesis of enhanced green fluorescent protein (EGFP) is achieved. Interestingly, EGFP synthesized in LM is entrapped in the HM, and the introduction of a cysteine residue to EGFP by genetic engineering further increases the amount of protein immobilization in the hydrogel matrices. These results suggest that the cell-free synthesis and HRP-catalyzed hydrogelation can be conducted in parallel in LM, and the eventual entrapment of the key components in HM is possible. Facile recovery of macromolecular products immobilized in HM by degrading the hydrogel network under reducing conditions should lead to the design of an easy-to-handle system to screen protein functions.
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