Integration of cell-free protein synthesis and purification in one microfluidic chip for on-demand production of recombinant protein.

Abstract

Recombinant proteins have shown several benefits compared with their non-recombinant counterparts in protein therapeutics. However, there are still some problems with the storage and distribution of recombinant proteins, owing to their temperature sensitivity. Microfluidic chips can integrate different functional modules into a single device because of the advantages of integration and miniaturization, which have the special potential to synthesize drugs when and where they are needed most. Here, we integrated cell-free protein synthesis and purification into a microfluidic chip for the production of recombinant protein. The chip consisted of a main channel and a branch channel. The main channel included two pinches, which were filled with template DNA-modified agarose microbeads and nickel ion-modified agarose beads as the cell-free protein synthesis unit and protein purification unit, respectively. The reaction mixture for protein synthesis was introduced into the main channel and first passed through the protein synthesis unit where the target protein was synthesized; next, the reaction mixture passed through the protein purification unit where the target protein was captured; and, finally, pure protein was collected at the outlet when washing buffer and eluting buffer were sequentially introduced into the branch channel. Enhanced green fluorescent protein (EGFP) was used as the model to investigate the performance of our chip. One chip could produce 70 μl of EGFP solution (144.3 μg/ml, 10.1 μg) per batch, and another round of protein synthesis and purification could be performed after replacing or regenerating nickel ion-modified agarose beads. It should be possible to produce other recombinant proteins on demand with this chip by simply replacing the template DNA.