A Convenient Self-Removing Affinity Tag Method for the Simple Purification of Tagless Recombinant Proteins.

In this work, we describe a novel self-cleaving affinity tag technology based on a highly modified split-intein cleaving element. In this system, which has recently been commercialized by Protein Capture Science, LLC under the name iCapTagTM , the N-terminal segment of an engineered split intein is covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong binding between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex. After unwanted impurities are washed from the resin, cleavage of the target protein is initiated by a shift of the buffer pH from 8.5 to 6.2. As a result, the highly purified tagless target protein is released from the column in the elution step. Alternately, the resin beads can be added directly to cell culture broth or lysate, allowing capture, purification and cleavage of the tagless target protein using a column-free format. These methods result in highly pure tagless target protein in a single step, and can thereby accelerate characterization and functional studies. In this work we demonstrate the single step purification of streptokinase, a fibrinolytic agent, and an engineered recombinant human hemoglobin 1.1 (rHb1.1). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression of high-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Basic Protocol 2: Purification of high-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform Alternate Protocol 1: Expression of low-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Alternate Protocol 2: Purification of low-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform.

Rugged bialkali photocathodes encapsulated with graphene and thin metal film.

Protection of free-electron sources has been technically challenging due to lack of materials that transmit electrons while preventing corrosive gas molecules. Two-dimensional materials uniquely possess both of required properties. Here, we report three orders of magnitude increase in active pressure and factor of two enhancement in the lifetime of high quantum efficiency (QE) bialkali photocathodes (cesium potassium antimonide (CsK2Sb)) by encapsulating them in graphene and thin nickel (Ni) film. The photoelectrons were extracted through the graphene protection layer in a reflection mode, and we achieved QE of ~ 0.17% at ~ 3.4 eV, 1/e lifetime of 188 h with average current of 8.6 nA under continuous illumination, and no decrease of QE at the pressure of as high as ~ 1 × 10-3 Pa. In comparison, the QE decreased drastically at 10-6 Pa for bare, non-protected CsK2Sb photocathodes and their 1/e lifetime under continuous illumination was ~ 48 h. We attributed the improvements to the gas impermeability and photoelectron transparency of graphene.

Photoemission from Bialkali Photocathodes through an Atomically Thin Protection Layer.

Photocathodes are essential components for various applications requiring photon-to-free-electron conversion, for example, high-sensitivity photodetectors and electron injectors for free-electron lasers. Alkali antimonide thin films are widely used as photocathode materials owing to their high quantum efficiency (QE) in the visible spectral range; however, their lifetime can be limited even in ultrahigh vacuum due to their high reactivity to residual gases and sensitivity to ion back-bombardment in these applications. An ambitious technical challenge is to extend the lifetime of bialkali photocathodes by coating them with suitable materials that can isolate the photocathode films from residual gases while still maintaining their highly emissive properties. We propose the use of graphene, an atomically thin two-dimensional material with gas impermeability, as a promising candidate for this purpose. Here, we report that high-quality bialkali antimonide can be grown on a two-layer (2L) suspended graphene substrate with a peak QE of 15%. More importantly, by comparing the photoemission through varying layers of graphene, we demonstrate that photoelectrons can transmit through few-layer graphene with a maximum QE of over 0.7% at 4.5 eV for 2L graphene, corresponding to a transmission efficiency of 5%. These results demonstrate important progress toward fully encapsulated bialkali photocathodes having both high QEs and long lifetimes using atomically thin protection layers.

Cesium-Coated Halide Perovskites as a Photocathode Material: Modeling Insights.

Photocathodes emit electrons when illuminated, a process utilized across many technologies. Cutting-edge applications require a set of operating conditions that are not met with current photocathode materials. Meanwhile, halide perovskites have been studied extensively and have shown a lot of promise for a wide variety of optoelectronic applications. Well-documented halide perovskite properties such as inexpensive growth techniques, improved carrier mobility, low trap density, and tunable direct band gaps make them promising candidates for next-generation photocathode materials. Here, we use density functional theory to explore the possible application of pure inorganic perovskites (CsPbBr3 and CsPbI3) as photocathodes. It is determined that the addition of a Cs coating improved the performance by lowering the work function anywhere between 1.5 and 3 eV depending on the material, crystal surface, and surface coverage. A phenomenological model, modified from that developed by Gyftopoulos and Levine, is used to predict the reduction in work function with Cs coverage. The results of this work aim to guide the further experimental development of Cs-coated halide perovskites for photocathode materials.

Highly efficient photoelectric effect in halide perovskites for regenerative electron sources.

Electron sources are a critical component in a wide range of applications such as electron-beam accelerator facilities, photomultipliers, and image intensifiers for night vision. We report efficient, regenerative and low-cost electron sources based on solution-processed halide perovskites thin films when they are excited with light with energy equal to or above their bandgap. We measure a quantum efficiency up to 2.2% and a lifetime of more than 25 h. Importantly, even after degradation, the electron emission can be completely regenerated to its maximum efficiency by deposition of a monolayer of Cs. The electron emission from halide perovskites can be tuned over the visible and ultraviolet spectrum, and operates at vacuum levels with pressures at least two-orders higher than in state-of-the-art semiconductor electron sources.

Angular scattering of protons through ultrathin graphene foils: Application for time-of-flight instrumentation.

Space plasma instruments often rely on ultrathin carbon foils for incident ion detection, time-of-flight (TOF) mass spectrometry, and ionization of energetic neutral atoms. Angular scattering and energy loss of ions or neutral atoms in the foil can degrade instrument performance, including sensitivity and mass resolution; thus, there is an ongoing effort to manufacture thinner foils. Using new 3-layer graphene foils manufactured at the Los Alamos National Laboratory, we demonstrate that these are the thinnest foils reported to date and discuss future testing required for application in space instrumentation. We characterize the angular scattering distribution for 3-30 keV protons through the foils, which is used as a proxy for the foil thickness. We show that these foils are ∼2.5-4.5 times thinner than the state-of-the-art carbon foils and ∼1.6 times thinner than other graphene foils described in the literature. We find that the inverse relationship between angular scattering and energy no longer holds, reaffirming that this may indicate a new domain of beam-foil interactions for ultrathin (few-layer) graphene foils.

Quantum Dot Thin-Films as Rugged, High-Performance Photocathodes.

Typical use of colloidal quantum dots (QDs) as bright, tunable phosphors in real applications relies on engineering of their surfaces to suppress the loss of excited carriers to surface trap states or to the surrounding medium. Here, we explore the utility of QDs in an application that actually exploits their propensity toward photoionization, namely within efficient and robust photocathodes for use in next-generation electron guns. In order to establish the relevance of QD films as photocathodes, we evaluate the efficiency of electron photoemission of films of a variety of compositions in a typical electron gun configuration. By quantifying photocurrent as a function of excitation photon energy, excitation intensity and pulse duration, we establish the role of hot electrons in photoemission within the multiphoton excitation regime. We also demonstrate the effect of QD structure and film deposition methods on efficiency, which suggests numerous pathways for further enhancements. Finally, we show that QD photocathodes offer superior efficiencies relative to standard copper cathodes and are robust against degradation under ambient conditions.