title: Refolding of difficult-to-fold proteins by a gradual decrease of denaturant using microfluidic chips.
abstract: Protein refolding is an important process to obtain active recombinant proteins from inclusion bodies (protein aggregates). However, the conventional refolding method of dialysis or dilution is a time consuming procedure and often, recovering yields of active proteins are low. In this study, we used controllable diffusion through laminar flow in microchannels to control the denaturant concentration. The performance of the designed microfluidic chips was evaluated by the refolding of difficult-to-fold proteins (citrate synthase and the zeta-associated protein 70-kDa protein kinase domain). We demonstrated this by varying the flow rates of the diluting buffer stream(s) and multi-junctions which could control the different flow rate ratios of the buffer stream(s) and the denatured protein stream. By this strategy, we were able to improve the efficiency of protein refolding. Our method achieved refolding within a short period of time at room temperature without the need of any small molecules or chaperone proteins. Moreover, the efficiency of protein refolding by microfluidic chip was found higher than that prepared by dialysis or dilution. These results suggest that microfluidic chips employing this strategy may provide miniaturized tools for rapid and efficient recovery of active proteins from inclusion bodies.
authors.affiliation: Nanotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, 807-1 Shuku, Tosu, Saga 841-0052, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Masaya
authors.initials: M
authors.lastname: Miyazaki
authors.forename: Maria Portia
authors.initials: MP
authors.lastname: Briones-Nagata
authors.forename: Hideaki
authors.initials: H
authors.lastname: Maeda
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D002950:Citrate (si)-Synthase / Q000737:chemistry*; D004058:Diffusion; D002479:Inclusion Bodies / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D002950:Citrate (si)-Synthase; D051746:ZAP-70 Protein-Tyrosine Kinase; C497262:Zap70 protein, mouse