A Cell Lysis and Protein Purification-Single Molecule Assay Devices for Evaluation of Genetically Engineered Proteins

摘要

We have developed two devices for evaluation of genetically engineered proteins in single molecule assay: on-chip cell lysis device, and protein purification-assay device. A motor protein, F_1-ATPase expressed in E. coli, was the focus of this report as the target protein. Cell lysis was simply performed by applying pulse voltage between Au electrodes patterned by photolithography, and its efficiency was determined by absorptiometry. The subsequent processes, purification and assay of extracted proteins, were applied to detect F_1-ATPase and to evaluate its activity. The specific bonding between his-tag in F_1-ATPase and Ni-NTA deposited on a glass surface was utilized for the purification process. After immobilization of F_1-ATPase, avidin-coated microspheres and adenosine triphosphate (ATP) solution were infused sequentially to assay the protein. Microsphere rotation was realized by activity of F_1-ATPase corresponding to ATP hydrolysis. Results show that the cell lysis device, under the optimum conditions, extracts a sufficient amount of protein for single molecule assay. Once cell lysate was injected to the purification-assay device, proteins were diffused in the lateral direction in a Y-shape microchannel. The gradient of protein concentration provides an optimal concentration for assay, that is, the highest density of rotating beads. Density of rotating beads is also affected by the initial concentration of protein injected into the device. The optimum concentration was achieved by our cell lysis device rather than by the conventional method using ultrasonic wave. Rotation speed was analyzed for several microspheres assayed in the purification-assay device, and the results were compatible with those of conventional bulk scale assay by the F_1-ATPase purification. In conclusion, we have demonstrated feasibility of on-chip cell lysis and assay for the sequential analysis without any pretreatment. It is expected that on-chip devices replacing conventional bioanalytical methods can be integrated in arntotal analysis system to evaluate engineered protein and DNA.