Light-Controlled Affinity Purification of Protein Complexes Exemplified by the Resting ZAP70 Interactome

Multiprotein complexes control the behavior of cells, such as of lymphocytes of the immune system. Methods to affinity purify protein complexes and to determine their interactome by mass spectrometry are thus widely used. One drawback of these methods is the presence of false positives. In fact, the elution of the protein of interest (POI) is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing, and 740 nm light for elution. Far-red light-induced elution is effective but very mild as the same buffer is used for the wash and elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the known interaction partners, and could filter out all other proteins

Cell-to-Cell-Variations of a Panasonic NCR18650B

Cell-to-Cell-Variations of Panasonic NCR18650B This dataset contains raw characterization data from 92 Panasonic NCR-18650B cells at the beginn of life. Information on the measurements performed can be taken in part from this readme file. Further details can be found in doi:\\ . Pulse measurements, pseudo-open circuit measurements, and impedance measurements are recorded. All measurements are performed in a Binder MK 155 climate chamber at 25°C. Pseudo Open Circuit Voltage POCV is recorded with a constant current of 0.05C in the charge and discharge direction. The measurements are performed with an Arbin LBT 5V-25A cell tester. The data is stored as a Matlab struct containing the following entries: pocv.ch % POCV of during charging pocv.dc % POCV of during discharging pocv.soc % SoC of the pocv curves The POCVs are interpolated to 5000 equidistant SOC points and sorted in the direction of the SOC vector. Impedance Impedance measurements are at 50% SOC(actual measured discharge C/20 capacity as reference) after 4h of relaxation. The measurements are conducted with a Zahner Zenium Pro spectroscope with an amplitude of 250 mA in a frequency range between 0.01 Hz and 10 kHz with 10 steps per decade. The data is stored as a Matlab struct containing the following entries: impedance.f % Frequency in Hz impedance.Z_imag % Imaginary Part of the impedance in Ohm impedance.Z_real % Real part of the impedance in Ohm impedance.Z % Complex valued impedance in Ohm Each row corresponds to a dataset of an individual cell and each column corresponds to a measurement frequency. The same applies to the .csv files. Pulse Test Pulse measurements are performed at 50% SOC (actual measured discharge C/20 capacity as reference) after 2h of relaxation. The measurements are performed with an Arbin LBT 5V-25A cell tester. The pulse test is performed with a current of 0.5C for 10s followed by a relaxation of 30 minutes. The discharge pulse follows the charge pulse. The data is stored as a Matlab struct containing the following entries: pulse.neg_time % Time of the discharge pulse sequence pulse.neg_voltage % Voltage of the discharge pulse sequence pulse.pos_time % Time of the charge pulse sequence pulse.pos_voltage % Voltage of the charge pulse sequenc

Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor

The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.</p

PSMA-Directed CAR T Cells Combined with Low-Dose Docetaxel Treatment Induce Tumor Regression in a Prostate Cancer Xenograft Model

While chimeric antigen receptor (CAR) T cell immunotherapy targeting CD19 has shown remarkable success in patients with lymphoid malignancies, the potency of CAR T cells in solid tumors is low so far. To improve the efficacy of CAR T cells targeting prostate carcinoma, we designed a novel CAR that recognizes a new epitope in the prostate-specific membrane antigen (PSMA) and established novel paradigms to apply CAR T cells in a preclinical prostate cancer model. In vitro characterization of the D7 single-chain antibody fragment-derived anti-PSMA CAR confirmed that the choice of the co-stimulatory domain is a major determinant of CART cell activation, differentiation, and exhaustion. In vivo, focal injections of the PSMA CAR T cells eradicated established human prostate cancer xenografts in a preclinical mouse model. Moreover, systemic intravenous CAR T cell application significantly inhibited tumor growth in combination with non-ablative low-dose docetaxel chemotherapy, while docetaxel or CAR T cell application alone was not effective. In conclusion, the focal application of D7-derived CAR T cells and their combination with chemotherapy represent promising immunotherapeutic avenues to treat local and advanced prostate cancer in the clinic

Spatiotemporally confined red light-controlled gene delivery at single-cell resolution using adeno-associated viral vectors

Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.ISSN:2375-254