Cell-free Protein Synthesis And Purification Based On Microfluidic Chips

Recombinant protein drugs have the advantages of high specificity and good biocompatibility.Its safety is significantly higher than that of small molecule drugs.Due to their temperature sensitivity,however,there are still some problems about storage and distribution of recombinant proteins.The production of single-dose of recombinant protein drugs on demand provides a solution for the problem.Microfluidic chips can integrate different functional modules on 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.Based on this,we designed and fabricated a microfluidic chip for the on-demand synthesis of recombinant proteins.The microchip integrated cell-free protein synthesis(CFPS)units and protein purification units.Specific research contents include:1.Linear template DNA-modified agarose beads were used for cell-free protein synthesisSince the linear template DNA is simpler to prepare than the plasmid DNA and can be designed flexibly,it can be prepared by PCR amplification and can be labeled according to different research purposes during the process of PCR amplification.Therefore,we prepared biotin-labelled linear template DNA by PCR amplification and modified it on streptavidin-coated agarose beads for CFPS to increase the local concentration of linear template DNA and increase protein yield.First,the non-labelled template DNA EGFP sequence was obtained by PCR amplification of the plasmid pET28a-EGFP;second,the template DNA was further amplified by using a 5'-end biotin-labelled forward primer and a non-labelled reverse primer to obtain biotinylated linear template DNA;finally,the biotinylated linear template was modified on streptavidin-coated agarose beads,which was used to synthesize protein in CFPS.The results showed that the template DNA-modified agarose beads could successfully synthesize enhanced green fluorescent protein(EGFP)in CFPS system.2.Integration of CFPS and protein purification in a single microfluidic chip for on-demand production of recombinant proteinBased on the previous research work,the microfluidic chip was combined with CFPS,and integrated a protein purification unit on the chip to achieve on-demand production of recombinant proteins.The chip consisted of a main channel and a branch channel: the main channel included two pinches,which filled with linear template DNA-modified agarose beads and nickel ion-modified agarose beads(Ni beads)as CFPS unit and protein purification unit,respectively.The reaction mixture for protein synthesis was introduced from the main channel and first passed through protein synthesis unit where the target protein was synthesized;next,the mixed solution passed through the protein purification unit,where the target protein was captured;at last,pure protein was collected at outlet when washing buffer and eluting buffer was sequentially introduced from the branch channel.We used enhanced green fluorescent protein(EGFP)as the model protein to investigate the performance of the chip.The results showed that one chip could obtain 144.3 ?g/mL EGFP at a time.Next round of protein synthesis and purification could be performed after replacing or regenerating Ni beads.This chip was expected to produce other recombinant proteins on demand only by replacing template DNA.3.The lable-free detection of kanamycin using cell-free protein synthesisKanamycin,a broad-spectrum antibiotic,is commonly used as a veterinary drug.However,excessive use will lead to its rediues in animal-derived foods,after intaking by human,kanamycin will interact with the 30 S subunit of ribosome,affecting the process of protein translation and causing poisoning.Based on this,this work used CFPS as a detection platform and GFP as a reporter protein to detect kanamycin.When kanamycin was present,the process of protein translation was affected,resulting in the yield of GFP reduced,that was,the fluorescence intensity decreased,thereby achieving the label-free detection of kanamycin in the range of 8-200 nmol/L,with a detection limit of 8 nmol/L and good selectivity.This method could be used to detect kanamycin in milk sample and showed great significance for the detection of excessive antibiotics addition in foods.