Sequential processing from cell lysis to protein assay on a chip enabling the optimization of an F(1)-ATPase single molecule assay condition.
We developed an integrated protein assay device, "Single Molecule MicroTAS (SMM)," which enables cell lysis, protein extraction, purification, and activity assay. The assay was achieved at the single-molecule scale for a genetically engineered protein, F(1)-ATPase, which is the smallest known rotary motor. A cell lysis condition, with a wide range of applied voltages (50-250 V) and other optimized values (pulse width: 50 micros; duty: 0.01%; electrode gap: 25 microm; total flow rate: 5 microL min(-1)) provided a high enough protein concentration for the assay. Successively, the protein was extracted and purified by specific binding in a microfluidic channel. During the assay process, the diffusion effect of lysate between a two-phase laminar flow contributes to optimizing the single-molecule assay condition, because the concentration of the original lysate from the E. coli solution is too high to assay. To achieve the most efficient assay condition, the protein diffusion effect on the assay was experimentally and numerically evaluated. The results reveal that, in our experimental conditions, concentrations of F(1) and other contaminated effluents are optimized for the F(1) rotational assay at a channel position. The adenosine triphosphate (ATP)-driven rotation speed measured in the SMM was compatible with that obtained by conventional purification and assay. Such a sequential process from cell lysis to assay proves that the SMM is an example of a sample-in-answer-out system for F(1) protein evaluation.
Nakayama
Tetsuya
T
Graduate School of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga, 525-8577, Japan.
Namura
Moriaki
M
Tabata
Kazuhito V
KV
Noji
Hiroyuki
H
Yokokawa
Ryuji
R
D001407:Bacillus / Q000201:enzymology; D001710:Biotin / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry*; D004058:Diffusion; D004566:Electrodes; D057075:Enzyme Assays / Q000295:instrumentation*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D008863:Microspheres; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism; D040081:Protein Array Analysis; D006180:Proton-Translocating ATPases / Q000032:analysis / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism; D013997:Time Factors
D002457:Cell Extracts; D009942:Organometallic Compounds; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D001710:Biotin; D019809:Streptavidin; D006180:Proton-Translocating ATPases; D009571:Nitrilotriacetic Acid