High-throughput system for the thermostability analysis of proteins.

Thermal stability of proteins is a primary metric for evaluating their physical properties. Although researchers attempted to predict it using machine learning frameworks, their performance has been dependent on the quality and quantity of published data. This is due to the technical limitation that thermodynamic characterization of protein denaturation by fluorescence or calorimetry in a high-throughput manner has been challenging. Obtaining a melting curve that derives solely from the target protein requires laborious purification, making it far from practical to prepare a hundred or more samples in a single workflow. Here, we aimed to overcome this throughput limitation by leveraging the high protein secretion efficacy of Brevibacillus and consecutive treatment with plate-scale purification methodologies. By handling the entire process of expression, purification, and analysis on a per-plate basis, we enabled the direct observation of protein denaturation in 384 samples within 4 days. To demonstrate a practical application of the system, we conducted a comprehensive analysis of 186 single mutants of a single-chain variable fragment of nivolumab, harvesting the melting temperature (Tm) ranging from -9.3 up to +10.8°C compared to the wild-type sequence. Our findings will allow for data-driven stabilization in protein design and streamlining the rational approaches.

Analysis of HepG2 cell response to a wide concentration range of mitomycin C using a multichannel quartz crystal microbalance system with a microscope.

The morphological response of HepG2 cells to mitomycin C was analyzed using a multichannel quartz crystal microbalance system equipped with a home-built movable microscope that enables the simultaneous acquisition of cell images and measurements of eight-channel quartz crystal microbalance. After 24 h of cell seeding, mitomycin C was injected into the culture medium. During the attachment process, the resonant frequency decreased, and the curves fitted well with the first-order lag response. Analysis of the response to mitomycin C revealed that the resonant frequency response curves varied with mitomycin C concentration. When the mitomycin C concentration was <10 µmol L-1, the delay time was observed before the increase in resonant frequency. When the mitomycin C concentration was extremely low, an additional decrease in resonant frequency was observed in the middle of the delay time that fitted well with the cumulative log-normal distribution curve. The resonant frequency response curves after the delay time fitted well with the cumulative log-normal distribution curves. The delay time and mean cumulative log-normal distribution time for the increase in resonant frequency correlated with the mitomycin C concentration; however, the mean time for the additional decrease in the resonant frequency did not show a statistically significant difference as a function of mitomycin C concentration. For mitomycin C concentrations of >20 µmol L-1, the response to the change in resonant frequency was rapid, and the response curves fitted well with the first-order lag response. The first-order lag response indicates that the response occurred simultaneously for all cells. The results showed that the time constant was independent of the tested mitomycin C concentration between 20 and 100 µmol L-1. These results suggested that different cell death processes occurred by mitomycin C. The findings of this study suggest that the system can be used to investigate cell death in adherent cells.

Comparison of cultured cell attachment on a temperature-responsive polymer, poly-L-lysine, and collagen using modeling curves and a thermal-controlled quartz crystal microbalance.

The characteristics of cultured cell attachment onto poly-L-lysine (PLL), collagen, and the thermoresponsive polymer poly(N-isopropylacrylamide) (PNIPAM) were studied using a quartz crystal microbalance (QCM). A QCM with microscope cameras enclosed in a Peltier chamber was developed to enable QCM measurements and microphotographic imaging to be conducted in a temperature-controlled CO2 incubator. Human hepatoma cell line HepG2 cells were cultured on the quartz crystals coated with PLL, collagen, and PNIPAM. Response curves of the resonant frequency of the quartz crystals during the cell attachment process were analyzed on the basis of the parameters of modeling curves fit to the experimentally obtained curves. Analysis of the fitting curves showed that the time constants of the first-lag response were 11 h for PLL, 16 h for collagen, and 38 h for PNIPAM and that the frequency change for the PNIPAM films was six times smaller than those for the PLL and collagen films. These findings were supported by photographic images showing wider cell spread on PLL and collagen than on PNIPAM. The response of cells on PNIPAM was measured during a thermal cycle from 37 to 20 °C to 37 °C. In the resonance frequency-resonance resistance (F-R) diagram, the slopes of ΔR/ΔF corresponding to the cell attachment process and those corresponding to the thermal cycling process differed; the positions in the F-R diagram also shifted to higher resonant frequencies after the thermal cycle. These results suggested that the mass effect decreased as a result of the weakening of the cell attachment strength by the thermal cycle because the molecular brushes of PNIPAM were disarranged.

Monitoring and Modeling of Living Cell Responses in the Attachment Process and Reaction to the Antitumor Reagent Cisplatin Studied by a Quartz Crystal Microbalance Combined with a Microscope.

The attachment process and response to an antitumor reagent for cultured cells were monitored with a quartz crystal microbalance (QCM) combined with a microscope. To fit the experimentally obtained curves of the resonant frequency, model equations of resonant frequency curves were built, and parameters of time constants and scale coefficients were determined. For the cell attachment process, a first-order lag response curve well fit the experimental curves. For the response to cisplatin, two response steps were observed in both QCM data and microscopic images, where the cells loosened in the first step and shrank in the second step. Resonant frequency responses for both processes were well fit by two logarithmic normal distribution functions. In addition, the dependence of the resonant frequency change on the cell number was also studied, and a cell-cell interaction model for attached cells was proposed to explain the saturation of the resonant frequency change in high density cell seeding.